Embryology - User contributions [en-gb]

Talk:2009 Group Project 3

2009-10-15T02:17:47Z

Z3218657: /* Log of Changes */

== Project Updates ==

Glossary (jo)

Referencing (sally)

Spell check/grammar (bronwyn)

Pictures - delete some, add relevant ones

research paper linkages - more maybe (everyone)

Format of overall page

Condensing text??? (possibly no condensing required)

====Log of Changes ====

First section has been proofed- Bronwyn

Glossary has been added - Jo P.S. Let me know if Ive missed some and I will add them

Have linked the current research and pictures with their respective Institutes and Departments- Jo

I've done the Referencing but could someone have a look and see if its ok. i made all the web pages links and movies into links - i think they would be better in the texts but i don't know where they should all go. if anyone has any ideas please move them! - Sal

GABY - would you be able to fix your images please - changing the nature articles (or cutting them so just the picture part is showing) and by making the bird, fish, frog images smaller! )

Copyright information added to the File:Development.jpg in accordance to request made by Mark Hill- Gaby

The bird, fish, frog images image has been made smaller by popular request and information pertaining to it has been added to the page of the picture so that it may only be accessed if the reader clicks onto the picture - Gaby

I did not change the articles as the fact that they were published at such a wide scale really impresses upon the reader the importance of the development of zebrafish as a model embryo and how widely doccumented it was- Gaby ???

PMIDs added to those references for which it was possible to find the PMIDs in response to the suggestion to fix up references- Gaby

Hey guys i just moved the image in the genome section -just down a bit so it didn't go directly under the image in the timeline.

Heey guys I've gone through most of the assignment and I think in terms of grammar its pretty good. Yay! -Bron

Current research adjusted into chronological order and also proofed - Jo

Additional copyright details included in current research pictures. Mutant embryo image was free to use and modify with the original author stated, as provided under the Creative Commons License - Jo

Just quickly cleaned up the websites shown throughout the page - good work guys werre done!

== Lab 10 ==

--[[User:Z3283499|Antonio Lee]] 10:55, 2 October 2009 (EST) Hi everyone, I will be working with you during the Lab10 Tutorial and here is the news link and PDF of the manuscript for your group exercise. I encourage you to read the paper before the tutorial. Also, please indicate next to the questions below (using either your initials or student number) which one of the four questions you wish to address.
:'''Group 3 :''' [http://www.myelomabeacon.com/news/2009/09/21/study-shows-mozobil-induces-mobilization-of-stem-cells-but-not-tumor-cells/ '''Study Shows Mozobil Induces Mobilization Of Stem Cells But Not Myeloma Tumor Cells'''] in The Myeloma Beacon Published online 21 September 2009 10:28 pm [[Media:ANAT2341_Lab10_2009_Group 3 Reading.pdf|Manuscript (PDF): Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization]]

:Question 1. What is the background to the existing problem / disease condition? 3220040
:Question 2. What approach / method did the research team take to tackle / improve the problem? 3218657
:Question 3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance? 3223194
:Question 4. What are the next steps in moving forward? What are the next or new hurdles to overcome? 3218792

complications can be stopping this proliferation of the cells and the spread of them. also, contamination can still occur by tumor cells.

Maybe this treatment can be applied to other stem cells other than bone in order to repair/ regenerate various tissue types

Another development may be using the proliferation of bone stem cells to use for treatment of other things than cancer

==Constructive Criticism of Coordinator==
--[[User:S8600021|Mark Hill]] 08:07, 8 October 2009 (EST) The following comments are general in nature in no specific order, as it would be inappropriate to suggest specific changes and then assess the final project. Comments will be added during this week and you still have one week before final submission.

* There is no list of changes made to your project on the basis of peer assessments.
* Background history is well covered.
* [[2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development|Timeline_and_Stages_of_Embryonic_Development]] [[:File:Mutant_embryo.png]]no source information for these images. [[:File:Development.jpg]] Copyright, are they able to be reproduced? I have made a major point about image sources.
* Images of development are always good (if you are allowed to use) but there is no description of developmental events?
* How does this model differ from others? What are the differences between fish and men?
* Referencing within your project seems inconsistent.
* Have you grasped why the zebrafish is a good model of vertebrate development?
* You need to provide a consistent "feel" as well, this can sometimes be difficult when several different people are editing a project. Overall the project still seems a little "word heavy" and has not organised the current research well.
* You need to accurately proof-read your text " the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm" makes no sense. It also suggests that you have not reviewed your work sufficiently.

==Constructive Criticism of Peers==

--[[User:Z3258567|Sando Rashed]] 09:06, 1 October 2009 (EST)hey good work with the page, lots of good pieces of information, i really like the timeline and stages of the embryonic development but you might want to add just a few words to each stage just to make the page suitable for all people to read (someone with no background will look at it and understand it), with your background info i like the idea but there isnt really a sentence structure, you might want to put it in dot form as that what is sounds like when reading it. you might want to add a glossary to the bottom of the page and add words that are unknown to people to make the reading more comprehensive. you might want to add some more photos to the current research just to make that part of the assignment a bit more appealing as there is a lot of information to grasp there. with your referencing, it is incorrent format you might want to go on the internet and look up harvard referencing (http://www.unisa.edu.au/ltu/students/study/referencing/harvard.pdf)but overall good job :D

--[[User:Z3218146|Julianna Lam]] 01:22, 1 October 2009 (EST)

- the history part has way too much information. it needs to be more concise.

-the images used for staging and timeline is awesome. set out very cleary and very easy to read and understand

- the mutation table is a very good idea although you can probably fix it up a bit so the information doesnt look as crammed up.

- great current research!

--[[User:Z3252231|Angama Yaquobi]] 20:48, 30 September 2009 (EST)

Congrats everyone, your team has done a great job. The project looks amazing.
*The info in the introduction is very informative especially under the heading of "Why Zebrafish".
*The images used in the timeline and stages of embryonic development is awesome and clicking on the period name gives alot of great information. It also makes the webpage look very nice and tidy- its structed very well.
*The section for genetics was very well informative especially comparing the zebrafish genome with human genome.
*I would recommend to add more images related to each section to make the webpage look more visually eye-catchy.
Overall it was a great effort by all the team members.well done.

--[[User:Z3186093|Jenny Guy]] 18:30, 30 September 2009 (EST)

Improvements:
* Some of your sentences sound as though you've cut and pasted them from websites. For example, do you guys really used "combing" in your vocabulary.
* The history goes more into finding out about the scientists instead of what they fully achieved. Try focus on that instead of the scientists preferences etc.
* Throughout the entire page there isn't much referencing at all! You'll have many paragraphs which dont refer to any article/text. You either need to include referencing in your text or at least place an number in reference to where you found out that information (corresponding with your bibliography).
* You need to reference your "websites" in the bibliography. By typing the url you are not referencing. Most of the articles are referenced so make sure you use the same system with the websites.
* You used youtube??? oh dear. I can understand you using it to gain an understanding but that cannot be used as a reference. seriously, anyone can upload it. So instead find out WHO uploaded it and find out if they published it on a RELIABLE website. Copyright?
* One of the outcomes Mark has asked for is a list of "links to related resources/research laboratories?" Where is this?

--[[User:Z3255007|Sadaf Masood]] 15:29, 30 September 2009 (EST)Hey Group 3! Congrats on your project guys, well done!
Ill just list down few things that i liked and what could be done better:

1. Needs glossary guys, so some of the scientific jargon can be understood easily

2. Your timeline is excellent, it has great pictures, maybe a little description along with it might make it more clear.

3. The project only has four sections, i m sure we need to do a bit about fertilization and detalied embryonic steps as well

4. References to specific articles are provided which is very informative if anyone wants to do an in-depth analysis.

5. Formatting is impressive and page layout is great!

overall, this is an awesome project which is simple and straight-forward. Great work guys! And Best of Luck!!!

--[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

Group 3 well done impression effort , resulting in a great formatted page, which summarises the requirements of the outcomes needed, it was straight to the point and briefly covered the main topic in regards to the zebrafish.

- The assignment has come together extremely well only a few minor edit here and there needed.

- The illustrations regarding (Timeline and Stages of Embryonic Development) are great, it extremely enhances the wiki page assignment allowing the readers to visualize the information which stand out in regard to the information it come along great.
The links to specfic research laboratories have been made throughout the information giving the reader additional information to learn more on the topic which is a great reference.

Suggestions to improve the assignment:

1. illustrations such as :"Development.jpg" are should be replace which more appropriate images which are more relevant with the topic and the illustrations should be more regarded in the information for the reader to relate and understand visually.

2. The Genetics information should include specfic genetic and chromosomal imagery revealing the structure of chromosome in comparison to humans and should identify why they are different.

3. Current research information should reveal a brief explanation on how zebrafish research has impacted human embryology and how it used and why it used to improve or help develop research programs.

4. Referencing is a problem, specfic referencing is needed as the university has guidelines. Visit this webpage I think it will help: www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

5. Glossary will also help the readers to understand specfic terms and make the assignment flow better.

Impressive wiki page, minor editing should be undertaken but overall the outcome were covered. --[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

--[[User:Z3217015|Mitchell Mathieson]] 09:42, 25 September 2009 (EST)Overall very very nice. Was nice and succinct, and easy to read. The information was relevant, and the current research interesting. Maybe the stages on different pages was a bit difficult to read; I would have liked to have seen at least a bit of information on the main page, which expands to more on your secondary page. The formatting of the references I think needs to be looked at maybe.

--[[User:Z3224449|Elide Newton]] 14:38, 26 September 2009 (EST) HELLO GROUP 3: Congratulations on a great assignment. Each section is equally proportioned, and well represented visually. My one point of constructive criticism would be to the section on current research. Your information is great, as it shows how the zebrafish is being used as a model for different areas of research. The one thing which would improve this would be to include the dates of these research papers, just so the reader can know how recent the work is. maybe introduce the research as Person et al (2009) has used the zebrafish to.... Also are there any pictures on this research? Just because research language is so technical, some pictures would break it up. Hope this helps you group 3.

--[[User:Z3126328|Jin Lee]] 16:06, 26 September 2009 (EST) congratulations to Group3! this is a really great assignment. overall, all the sections are well represented visually.
I liked the history part mostly! easy to read! Maybe the current research section can be improved by adding some images and relevant links. I found zebrafish is very interesting!thank you

--[[User:Z3187802|Vishnnu Shanmugam]] 19:32, 26 September 2009 (EST)Well done Zebra fish group. You've put together a well balanced assignment. The images under "Timeline and Stages of Embryonic Development" are impressive, really make the assignment stand out and the text flows nicely. I also noticed the links to researchers and research laboratories have been made throughout the text and because they are under specific headings, the reader will know what the information in the link will focus on. Additions to improve the assignment:

- there is some irrelevant images in the the text; the images "A 1981 issue of Nature journal" & "Development.jpg" are Unnecessary and could be replaced with others that support the text or summarize complex processes. (eg. motor neuron development in zebrafish or a time line showing the evolution in the use of zebrafish). The second image "Development.jpg" is better replaced with the image "A Zebrafish Pigment Mutant" as it is relevant to the text. The current research does not mension or explain the Zebrafish Pigment Mutant and thus is Unnecessary there.

- The Genetics section could do with an image of zebrafish chromosomes. This can be a simple hand drawn diagram and can be compared to the human chromosomal makeup.

- The current research section could use a description of how zebrafish research has impacted human embryology.

-The assignment needs to be properly referenced. see www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

- A Glossary would also complement the text.

Overall very impressive, only needs minor editing.

-[[User:Z3252340|Emily Wong]] 11:04, 27 September 2009 (EST) To begin with, you have done a great job group 3. It is a well researched, structured and organized page. The student contribution to the work is fairly even. The condensation of the written content is done well, with the combining of the timeline and stages in one section and extensive use of visual representation. However, information is not provided when clicking on the pictures as to what is in them. A description or explanation of what is occurring in each stage or at each time point would improve this project. Some of the images used have not been referenced properly in the image pages. Referencing is good and an extensive reference list has been provided. The content provided is very detailed by still brief and relevant to each section. This project could also be improved by adding some examples of current research and also referencing information throughout the written content. Perhaps an inclusion of a Pubmed search link in the current research section would be a good idea so that viewers can see what other research has been done using the zebrafish.

--[[User:Z3126345|Gang Liu]] 16:17, 27 September 2009 (EST)This is a very informative, well constructed and concise wikipage. It has demonstrated consistency throughout the page. There are a number of major subheadings have been included history, timeline, development, genetics and current embryology research as well as hand drawing. In addition, it has demonstrated extended research of literatures. For example, by dividing the history section in a number of subheadings such as "in the beginning", "Charles Kimmel", "Hesitations", etc. This has indicates the in-depth understanding of zebrafish embryo model use. It allows the reader to appreciate story behind the experiment, as well as scientists' thinking process and thoughts. "...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work...".

The graphic delivery of timeline and stages are very self-explanatory and powerful. A picture explains a thousand words. It makes the section easy to follow, and interesting to read also.

However, this project can be improved by considering the following points.

*I found genetic and current embryology section were very lengthy and wordy, and lack of pictures. In addition, referencing in these two sections were inconsistent with previous sections;

*Lack of glossary list. Such as "oxidative phosphrylation", "N-ethyl-N-nitrosourea (ENU)", "Diploid" and "Haploid" need to provide meanings;

*Inconsistent referencing. Name of author need to keep in alphabetical order. Also articles and web based materials are mixed. The first five references were inconsistent with the rest in style;

Last few words. I enjoyed reading it, learned something out of it. Big thumb up for me.

--[[User:Z3217686|Thomas Dangerfield]] 13:54, 28 September 2009 (EST)Well done guys! Nice, visually stimulating assignment! Just a few points about possibly having a little more written info on the timeline, just to help explain what is in each image. Also some pictures of the chromosomes of the zebrafish would be a handy visual tool. This may just be a little picky, but i notice that you have a few heading issues:

- Beginning not Begining. (In the begining..)

- Genetics of the Zebrafish and Embryology, and the you have a 'Genetics and Embryology' a couple of headings down which seems a little unnecessary.

Just as a hint on being thorough, maybe a glossary could have been included, as well as formatting for your references. Still, a well presented assignment has been done here!

--[[User:Z3185685|Sumaiya Rahman]] 16:06, 28 September 2009 (EST) Hey guys. Well done on the assignment overall. It was interesting to read about the zebrafish. The page was well introduced. It is good that you broke up the information using subheadings. This makes the page a lot easier to read as it breaks up the big slabs of text. I think it has been mentioned, but there are a couple of spelling mistakes. Also the sentence “This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work” needs to be fixed up. This is only a minor error. The history section could look a little bit less messy if the pictures were placed on the same side. “The fish is a frog... is a chicken... is a mouse” picture is very cute! But maybe a little bit too big because we need to scroll across to see it (or is that just me?). You have some great references and links and a great timetable and stages section with good visuals. The genetics and current research sections have a lot of text, maybe finding a way of making these sections more visually appealing would help the readers. Also including a glossary would help. Overall nice work!

--[[User:Z3215682|Carly Mooney]] 11:43, 29 September 2009 (EST)
Impressive, it looks as though much time and effort went into making the page easy to read yet informative.A few suggestions:
*some of the images I thought weren't entirely relevant, e.g.A 1981 issue of Nature journal" & "Development.jpg". maybe even crop it so that it is just the covers photograph in the image, rather than the entire journal.
*Maybe decrease the size of the fish is a frog is a chicken is a mouse jpg
*Maybe move the genetics picture further down as it is very similar and close to the timeline and stages section
*I think a glossary may help
*The references to be referenced properly, and the articles should have the pubmed number with their reference
*a few grammatical errors that need to be fixed up
These are all minor thoughts, well done.

--[[User:Z3254857|Begum Sonmez]] 14:53, 30 September 2009 (EST)
Hey Group 3. Let me start off by saying that I found your project on the Zebrafish unique. Under the history section:
*There were some sentences that were unnessesary such as 'Streisinger was not the only scientist working on the embryologic development of the zebrafish.'

*It was different and iteresting to learn about the different views of Streisinger and Kimmel. I enjoyed that fact that the structure wasn't entirely date than author than finding, but the inclussion of 'disputes and uncertainties' ('...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with.') as well.

*The information flows from Streisinger to Kimmel to hesitations.

*There is too much text bunched up under the sub-headings 'Taking the plunge', 'The Big Screen', and 'The Importance of Mutations'. Do not delete any text because I found it very interesting, but I suggest the use of Sub-sub headings, or some dot points. Another example would be to have the sub-heading as 'The Big Screen (1993-1996)'. This gets rid of some text.

The only thing that I found lacking under the Timeline/Stages section was a description of the image. I don't think a detailed descritption is necessary because the table is very well constructed. For example, state when the heart begins to develop or when it has finished developing, and place this information on the related image's page.

One of my favourite parts was the table under the Genetics section 'Mutations in Zebrafish Causing Developmental Heart Defects ' because the information is well constructed into a table format in a way that makes sense. In other words, the information is very clear. The information under 'How are mutations achieved?' was very interesting, I never before exactly thought about different ways of mutating.

I found a little problem in the first paragraph of current research section: 'Although the embryo was '''consistently''' studied '''continuously''' over the years...'. Just remove either bolded word. Some things that I think might enhance this section would be to include:
*Some links to published articles dealing with recent experimental research.
*Dates to identify when the research by The Laboratory of Molecular Genetics and Developmental Biology (China) was undertaken.
*The names of people/labortaories involved on the experiments on the gene of lissencephaly LIS1. In other words, inlude a specific example of a certain group of researchers that have utilised this gene.

And finally, the video under 'eye disorders' was interesting, relevant and informative at the same time. I liked it very much. Good work Group 3.

----

--[[User:S8600021|Mark Hill]] 01:44, 8 September 2009 (EST) A single image of a fish, well that really gets across the message of zebrafish development, interpret the information you have read and put it into a format that will interest the reader.

ZebraFish - Zebrafish are really cool...

I'm happy with zebrafish... never heard of them but they sound interesting :) what does everyone else think? ...Gaby Pinget

Great, who else is in our group then... Oh and i'm Sal by the way

Ok so i'm not sure who the other two are because i don't know your number but just for reference my number is z3218657.
I found this cool website with some pictures of Zebrafish embryo's developing... its pretty cool...

http://www.cas.vanderbilt.edu/bioimages/animals/danrer/zfish-devel.htm

and this website shows all the stages and times and such good for a timeline

http://zfin.org/zf_info/zfbook/stages/stages.html

Research Topics for this week:
- Timeline and Staging (Sal)
- History of Model Use (Gaby)
- Genetics (Bronwyn)
- Current Embryology Research (Jo)
--> Share what we've learnt with the group next session.

Hey Guys I have a lot of info... and i don't think i should post it all up here because there is a lot of stuff... Mainly all about the Staging though. I am going to put up a summary of the different stages. The website i gave you before ZFIN is the zebrafish database of model organisms. its great!

'''Zygote Period''' – Lasts for 0-0.75hours
Transition of one cell to two cells.
Fertilisation occurs activating cytoplastimic movement. The animal poles within the cell segregate the blastodisc from the yolk cytoplasm. Segregation continues into the cleavage staging.

'''Cleavage Period''' – Lasts for 0.75-2.25 hours
Transition from two cells to 128 cells
After the first cleavage the blastocysts division is approx every 15 minutes.

'''Blastula Period''' – Lasts for 2.25-5.25 hours
Transition from 128 cells to 50% epibolby
“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – Direct quote from the ZFIN website

'''Gastrula Period''' – Lasts for 5.25-10.33 hours
Transition from 50% epibolby to 1-4 somites
The gastrula period ends when epiboly is complete, and the tail bud has formed. Here each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations.

'''Segmentation period''' – Lasts from 10.33 – 24 hours
Transistion from 1-4 somites to Prim-5
Here dermis, vertebrae and skeletal muscle are formed

'''Pharyngula Period''' – Lasts from 24 – 48 hours
Transition from Prim 5 to Long-pec
The body axis begins to straighten and the fins begin to develop.

http://www.youtube.com/watch?v=0hGT667ktTw

'''Hatching Peroid''' – Last from 48 to 72hours
Transitions from Long-Pec to Protruding-mouth
In this period, primary organ systems develop and cartilage development begins.

'''Larval Period''' – Lasts from 72hours to 30days
transition from Protruding-mouth to Day 30-44
The pectoral fin continues to develop and the internal organs become more complex. Development continues.

'''Juvenille Period''' – Lasts from 30-44 days
Here adult fins and pigments as well as 12 teeth develop.

'''AdultPeriod''' – Lasts90days to 2 years
Full Breeding Adult.

http://www.youtube.com/watch?v=5ygcu9BRXI0 - Zebrafish heart beating!

http://www.youtube.com/watch?v=TbErcmhzUSY - alcohol effects on Zebrafish embryo

Websites used
http://dev.biologists.org/cgi/content/abstract/dev.022673v1

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

Hey! This is a really good site with a bunch of links relating to the different areas we have to research:
http://www.sanger.ac.uk/modelorgs/zebrafish.shtml

Hey guys.... Ive found a really good picture of the zebrafish embryo development (like the human one) but I dont know how to put it up, was thinking it might be better if it was actually on the page rather than a link to get the picture. JO

well I think that I just uploaded a picture but I have no idea where to... wow I'm so lost... Maybe that's a good thing because I have a feeling that it was copy right protected... DAMN COPY RIGHT ah ha ha

Hey all, all the websites that i have put up on this page have really good images... I have emailed the three of them and asked if it was ok if i used them to put up on here and i am awaiting a reply so
hopefully they will let us use them. Also i am having a tonne and a half of trouble uploading a InDesign Timeline image i created of the timeline and also having some issues with the net so i might
bring them to class and see if the computers there are readable other wise i am screwed and will just have to start all over again. I have a more complete Timeline and Staging format that i have
created as well. I'll put that up when i try and upload the image again. Peace. - Sal

Hey Hey So I got permission from Judy Cebra-Thomas to use all her images on her website which are each stage of development - YAY we just have to accredit them. So today apart from working on
unwinding my InDesign TImeline and putting the info up in bullets i am going to start to figure out whats happening in all the images and try an upload them onto the site so hopefully tomorrow we
will have an almost finished timeline and staging section! - Sal

Right so I've posted a link to the current sequencing status on the genetics part of our site. This updates pretty much everyday so I will be able to update ours right before submission so we have up-to-date info. This is however a minor detail so I'm writing it here to help us all remember! :) thanks

'''Hey guys!! please read this: I put up pictures which are really just to make it look pretty rather than add any info. Can you please tell me if you think they're stupid or not serious enough? I promise that I wont be offended! I understand if any of you think that we should take our assignment more seriously than that... it's just hard to find images for a history of zebrafish use!'''

I dont really get the light bulb lol. Sally were u able to get the embryo stage images as a link to the info as u wanted???? I think its a really good idea if we could do it, i think it might be a matter of asking someone who's actually good with computers to do it lol. Im still complying info for the current research section, currently in qld but i wanna have it up hopefully by this week, at least part of it anyways. Im gonna try n get some pictures but every place that ive asked think im an actual doctor lol so its been a bit hard.

Ok so not quite sure how to do the linking but i might just make it all link off the page into separate little pages... does that make sense??? Probably not but i can try explain it better on thursday. The
images take me 1.5hours each to upload because i have to make the image in indesign, Grab it and then transform the image to a PNG file as that is one that this website allows for uploading! So they
are coming up but ever so slowly! When i got permission to use the images i just told them I was a student studying Advance Science from UNSW. I was enrolled in an embryology course there where
we were doing an assignment on the Embryology of Zebrafish. Could i please use your images with referencing and a link to your webpage or document. Thats kind of what i said! i'm hoping to have it
all up and ready on thursday but might have to finish it friday night as have friends down from QLD that tend to make my place messy and have a house inspection on that ARVO!!! - Cheers SAL

Hey Sally, just so u know. I moved the websites you used to the references section so it didnt seem odd to keep it after ur info.

Hey Sally, we were thinkin about whether or not we could mix the timeline with the stages, so we thought that maybe if on the actual page we had the timeline with the pictures, then by clicking on the pictures there would be the info that u got or maybe even putting it all into a table so the pictures & info are more integrated.

Hey guys sorry i wasn't in class yesterday i had a funeral. What i was thinking of doing was actually moving all the data off the page and you click on it to go to another page which as all the info! but
the idea of clicking on the picture and going to it sounds great. One flaw is that is it ok if i use the pictures twice because i have been cutting, altering, adding text and flattening the images so that
they have a caption of what is happening. If i just cut another image to show each stage and then you click on that to go to it i think that could be cool as long as when you open the next page it still
has the information and the image with the caption of what is occurring! Hope thats ok. I will work on it all tonight and hopefully finish. Its taking a lot more time than expect sorry!Sal

hey Guys so I don't know if you guys have checked the website out yet but i have been working on it today! I continued what i was doing with the linked page and have also drawn my own diagrams
which are colour coded and easy for people to understand what is going on during the stages. I thought it looked cool. I haven't finished yet - a few more periods to go but i can't look at the screen
any more so i will do some on tuesday night! Hope thats ok. Just wondering though about the Nature article images above it kind of comes across the screen and i was wondering if we could somehow
move the image a little higher or enter down some spaces so that the timelines section is lower and the nature article doesn't come over the images pushing the table to the left! Let me know what you
all think. Sal

Hey Sal, your part of the project looks great! I really like the idea of clicking onto another page to get the info. works really really well and it's really easy to understand. As for the size of the picture obscuring your section, I think that you already thumbnailed it and it looks fine. Also, do you know how to put a youtube video onto the webpage? I was able to for a project in another class and it looked good but that was much easier because it had a link to a bunch of plug ins i could use. If anyone has any ideas please let me know! Gaby

Hey Sally, ur part looks awesome!!! Everyone done forget about your references...ive tried making the ones there so far look like it was done by one person lol. I dont know how to do the youtube clip thing but i have one too, was thinking maybe we should have a section for helpful links or do u think we should just put the links in with watever section its assiciated with. Wat do u guys think??? Jo

Hmm I was just thinking that the best thing to do was to put up the links strait after whatever they're associated with, that way they are supplimentary to what the reader is looking at rather than random extras at the end. That'll prob work best, right? Gaby

Thanks guys I've put a tonne of effort in so hope it pays off! I was going to fix up the references (cause they just have my websites without being real references) but it took me a lot longer than i thought for the images as you
can see its 5am. Nice. Yes i think linking the videos with the relevant parts is best. I have added links to videos throughout my section. Also I have made it so that when you put your mouse over the images it comes up with its own
reference/copy right info. Also when adding a link to another website its best to name the website instead of having just the web address. Its really easy if you don't know how and i can show you how to fix them up. When we get our
feedback from the other groups we can add and so forth before the project is actually marked which is really great. I think we need more info with the Genetics part but i figure bron is doing that tomorrow! Jo I really love your
sections pics and info it great and to the point!Gaby, yeah i did move it hope thats ok. I do have one question though and not meaning to hurt your feelings but i'm not that keen on the drawn image of the bird,fish,frog and mouse.
It doesn't really fit. Sorry, I mean obviously the decision is up to you, but i think it just looks a bit odd in the middle of the page because it you see it first and think , what? That was just some of the other feedback i got
from some of my friends that i asked to check out the website and see how they could maneuver around it. So that's my major spiel. Catch ya tomorrow. Sal

2009 Group Project 3

2009-10-15T02:14:30Z

Z3218657: /* Genetics of the ZebraFish and Embryology */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Beginning... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organism and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed mapping out of genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]

===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae. By the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

[[File:A fish is a frog is a chicken is a mouse.jpg|thumbnail|left]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combining these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the left half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

[http://www.sanger.ac.uk/Projects/D_rerio/ Current Sequencing Status]

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, follow the link to [http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml The Sanger Trust institute Website]

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years from 1980s to 2009. Although the embryo was consistently studied over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fight cancer, to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understanding about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. These studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were however maintained as plastic phenotypes, expressing specific cell types including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology (MIT), Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence MIT determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Genetic Mutations Associated with Sight===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contributed to maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for [http://www.youtube.com/watch?v=ZItgyfuxsfM eye disorder research]

===Significance of Notochord===
One of the most significant characteristics of the embryo is the notochord; a midline structure required for skeletal development of the vertebrae and is the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Brain and Developmental Deficiencies===
The zebrafish has been utilized to express the gene of lissencephaly LIS1; a severe disease associated to brain malformation such as Miller-Dieker syndrome. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development within mature tissue samples. The LIS1 gene was predominately evident within brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. It was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149. PMID: 15138510

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967. PMID: 11691861

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513. PMID: 12468254

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724. PMID: 12209146

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-15T02:13:58Z

Z3218657: /* Danio rerio Genome Sequencing Project */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Beginning... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organism and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed mapping out of genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]

===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae. By the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

[[File:A fish is a frog is a chicken is a mouse.jpg|thumbnail|left]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combining these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the left half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, follow the link to [http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml The Sanger Trust institute Website]

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years from 1980s to 2009. Although the embryo was consistently studied over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fight cancer, to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understanding about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. These studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were however maintained as plastic phenotypes, expressing specific cell types including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology (MIT), Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence MIT determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Genetic Mutations Associated with Sight===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contributed to maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for [http://www.youtube.com/watch?v=ZItgyfuxsfM eye disorder research]

===Significance of Notochord===
One of the most significant characteristics of the embryo is the notochord; a midline structure required for skeletal development of the vertebrae and is the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Brain and Developmental Deficiencies===
The zebrafish has been utilized to express the gene of lissencephaly LIS1; a severe disease associated to brain malformation such as Miller-Dieker syndrome. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development within mature tissue samples. The LIS1 gene was predominately evident within brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. It was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149. PMID: 15138510

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967. PMID: 11691861

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513. PMID: 12468254

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724. PMID: 12209146

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-15T02:11:24Z

Z3218657: /* Genetic Mutations Associated with Sight */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Beginning... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organism and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed mapping out of genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]

===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae. By the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

[[File:A fish is a frog is a chicken is a mouse.jpg|thumbnail|left]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combining these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the left half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years from 1980s to 2009. Although the embryo was consistently studied over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fight cancer, to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understanding about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. These studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were however maintained as plastic phenotypes, expressing specific cell types including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology (MIT), Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence MIT determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Genetic Mutations Associated with Sight===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contributed to maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for [http://www.youtube.com/watch?v=ZItgyfuxsfM eye disorder research]

===Significance of Notochord===
One of the most significant characteristics of the embryo is the notochord; a midline structure required for skeletal development of the vertebrae and is the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Brain and Developmental Deficiencies===
The zebrafish has been utilized to express the gene of lissencephaly LIS1; a severe disease associated to brain malformation such as Miller-Dieker syndrome. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development within mature tissue samples. The LIS1 gene was predominately evident within brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. It was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149. PMID: 15138510

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967. PMID: 11691861

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513. PMID: 12468254

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724. PMID: 12209146

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

Talk:2009 Group Project 3

2009-10-14T23:09:18Z

Z3218657: /* Log of Changes */

== Project Updates ==

Glossary (jo)

Referencing (sally)

Spell check/grammar (bronwyn)

Pictures - delete some, add relevant ones

research paper linkages - more maybe (everyone)

Format of overall page

Condensing text??? (possibly no condensing required)

====Log of Changes ====

First section has been proofed- Bronwyn

Glossary has been added - Jo P.S. Let me know if Ive missed some and I will add them

Have linked the current research and pictures with their respective Institutes and Departments- Jo

I've done the Referencing but could someone have a look and see if its ok. i made all the web pages links and movies into links - i think they would be better in the texts but i don't know where they should all go. if anyone has any ideas please move them! - Sal

GABY - would you be able to fix your images please - changing the nature articles (or cutting them so just the picture part is showing) and by making the bird, fish, frog images smaller! )

Copyright information added to the File:Development.jpg in accordance to request made by Mark Hill- Gaby

The bird, fish, frog images image has been made smaller by popular request and information pertaining to it has been added to the page of the picture so that it may only be accessed if the reader clicks onto the picture - Gaby

I did not change the articles as the fact that they were published at such a wide scale really impresses upon the reader the importance of the development of zebrafish as a model embryo and how widely doccumented it was- Gaby ???

PMIDs added to those references for which it was possible to find the PMIDs in response to the suggestion to fix up references- Gaby

Hey guys i just moved the image in the genome section -just down a bit so it didn't go directly under the image in the timeline.

Current research adjusted into chronological order and also proofed - Jo

Additional copyright details included in current research pictures. Mutant embryo image was free to use and modify with the original author stated, as provided under the Creative Commons License - Jo

== Lab 10 ==

--[[User:Z3283499|Antonio Lee]] 10:55, 2 October 2009 (EST) Hi everyone, I will be working with you during the Lab10 Tutorial and here is the news link and PDF of the manuscript for your group exercise. I encourage you to read the paper before the tutorial. Also, please indicate next to the questions below (using either your initials or student number) which one of the four questions you wish to address.
:'''Group 3 :''' [http://www.myelomabeacon.com/news/2009/09/21/study-shows-mozobil-induces-mobilization-of-stem-cells-but-not-tumor-cells/ '''Study Shows Mozobil Induces Mobilization Of Stem Cells But Not Myeloma Tumor Cells'''] in The Myeloma Beacon Published online 21 September 2009 10:28 pm [[Media:ANAT2341_Lab10_2009_Group 3 Reading.pdf|Manuscript (PDF): Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization]]

:Question 1. What is the background to the existing problem / disease condition? 3220040
:Question 2. What approach / method did the research team take to tackle / improve the problem? 3218657
:Question 3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance? 3223194
:Question 4. What are the next steps in moving forward? What are the next or new hurdles to overcome? 3218792

complications can be stopping this proliferation of the cells and the spread of them. also, contamination can still occur by tumor cells.

Maybe this treatment can be applied to other stem cells other than bone in order to repair/ regenerate various tissue types

Another development may be using the proliferation of bone stem cells to use for treatment of other things than cancer

==Constructive Criticism of Coordinator==
--[[User:S8600021|Mark Hill]] 08:07, 8 October 2009 (EST) The following comments are general in nature in no specific order, as it would be inappropriate to suggest specific changes and then assess the final project. Comments will be added during this week and you still have one week before final submission.

* There is no list of changes made to your project on the basis of peer assessments.
* Background history is well covered.
* [[2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development|Timeline_and_Stages_of_Embryonic_Development]] [[:File:Mutant_embryo.png]]no source information for these images. [[:File:Development.jpg]] Copyright, are they able to be reproduced? I have made a major point about image sources.
* Images of development are always good (if you are allowed to use) but there is no description of developmental events?
* How does this model differ from others? What are the differences between fish and men?
* Referencing within your project seems inconsistent.
* Have you grasped why the zebrafish is a good model of vertebrate development?
* You need to provide a consistent "feel" as well, this can sometimes be difficult when several different people are editing a project. Overall the project still seems a little "word heavy" and has not organised the current research well.
* You need to accurately proof-read your text " the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm" makes no sense. It also suggests that you have not reviewed your work sufficiently.

==Constructive Criticism of Peers==

--[[User:Z3258567|Sando Rashed]] 09:06, 1 October 2009 (EST)hey good work with the page, lots of good pieces of information, i really like the timeline and stages of the embryonic development but you might want to add just a few words to each stage just to make the page suitable for all people to read (someone with no background will look at it and understand it), with your background info i like the idea but there isnt really a sentence structure, you might want to put it in dot form as that what is sounds like when reading it. you might want to add a glossary to the bottom of the page and add words that are unknown to people to make the reading more comprehensive. you might want to add some more photos to the current research just to make that part of the assignment a bit more appealing as there is a lot of information to grasp there. with your referencing, it is incorrent format you might want to go on the internet and look up harvard referencing (http://www.unisa.edu.au/ltu/students/study/referencing/harvard.pdf)but overall good job :D

--[[User:Z3218146|Julianna Lam]] 01:22, 1 October 2009 (EST)

- the history part has way too much information. it needs to be more concise.

-the images used for staging and timeline is awesome. set out very cleary and very easy to read and understand

- the mutation table is a very good idea although you can probably fix it up a bit so the information doesnt look as crammed up.

- great current research!

--[[User:Z3252231|Angama Yaquobi]] 20:48, 30 September 2009 (EST)

Congrats everyone, your team has done a great job. The project looks amazing.
*The info in the introduction is very informative especially under the heading of "Why Zebrafish".
*The images used in the timeline and stages of embryonic development is awesome and clicking on the period name gives alot of great information. It also makes the webpage look very nice and tidy- its structed very well.
*The section for genetics was very well informative especially comparing the zebrafish genome with human genome.
*I would recommend to add more images related to each section to make the webpage look more visually eye-catchy.
Overall it was a great effort by all the team members.well done.

--[[User:Z3186093|Jenny Guy]] 18:30, 30 September 2009 (EST)

Improvements:
* Some of your sentences sound as though you've cut and pasted them from websites. For example, do you guys really used "combing" in your vocabulary.
* The history goes more into finding out about the scientists instead of what they fully achieved. Try focus on that instead of the scientists preferences etc.
* Throughout the entire page there isn't much referencing at all! You'll have many paragraphs which dont refer to any article/text. You either need to include referencing in your text or at least place an number in reference to where you found out that information (corresponding with your bibliography).
* You need to reference your "websites" in the bibliography. By typing the url you are not referencing. Most of the articles are referenced so make sure you use the same system with the websites.
* You used youtube??? oh dear. I can understand you using it to gain an understanding but that cannot be used as a reference. seriously, anyone can upload it. So instead find out WHO uploaded it and find out if they published it on a RELIABLE website. Copyright?
* One of the outcomes Mark has asked for is a list of "links to related resources/research laboratories?" Where is this?

--[[User:Z3255007|Sadaf Masood]] 15:29, 30 September 2009 (EST)Hey Group 3! Congrats on your project guys, well done!
Ill just list down few things that i liked and what could be done better:

1. Needs glossary guys, so some of the scientific jargon can be understood easily

2. Your timeline is excellent, it has great pictures, maybe a little description along with it might make it more clear.

3. The project only has four sections, i m sure we need to do a bit about fertilization and detalied embryonic steps as well

4. References to specific articles are provided which is very informative if anyone wants to do an in-depth analysis.

5. Formatting is impressive and page layout is great!

overall, this is an awesome project which is simple and straight-forward. Great work guys! And Best of Luck!!!

--[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

Group 3 well done impression effort , resulting in a great formatted page, which summarises the requirements of the outcomes needed, it was straight to the point and briefly covered the main topic in regards to the zebrafish.

- The assignment has come together extremely well only a few minor edit here and there needed.

- The illustrations regarding (Timeline and Stages of Embryonic Development) are great, it extremely enhances the wiki page assignment allowing the readers to visualize the information which stand out in regard to the information it come along great.
The links to specfic research laboratories have been made throughout the information giving the reader additional information to learn more on the topic which is a great reference.

Suggestions to improve the assignment:

1. illustrations such as :"Development.jpg" are should be replace which more appropriate images which are more relevant with the topic and the illustrations should be more regarded in the information for the reader to relate and understand visually.

2. The Genetics information should include specfic genetic and chromosomal imagery revealing the structure of chromosome in comparison to humans and should identify why they are different.

3. Current research information should reveal a brief explanation on how zebrafish research has impacted human embryology and how it used and why it used to improve or help develop research programs.

4. Referencing is a problem, specfic referencing is needed as the university has guidelines. Visit this webpage I think it will help: www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

5. Glossary will also help the readers to understand specfic terms and make the assignment flow better.

Impressive wiki page, minor editing should be undertaken but overall the outcome were covered. --[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

--[[User:Z3217015|Mitchell Mathieson]] 09:42, 25 September 2009 (EST)Overall very very nice. Was nice and succinct, and easy to read. The information was relevant, and the current research interesting. Maybe the stages on different pages was a bit difficult to read; I would have liked to have seen at least a bit of information on the main page, which expands to more on your secondary page. The formatting of the references I think needs to be looked at maybe.

--[[User:Z3224449|Elide Newton]] 14:38, 26 September 2009 (EST) HELLO GROUP 3: Congratulations on a great assignment. Each section is equally proportioned, and well represented visually. My one point of constructive criticism would be to the section on current research. Your information is great, as it shows how the zebrafish is being used as a model for different areas of research. The one thing which would improve this would be to include the dates of these research papers, just so the reader can know how recent the work is. maybe introduce the research as Person et al (2009) has used the zebrafish to.... Also are there any pictures on this research? Just because research language is so technical, some pictures would break it up. Hope this helps you group 3.

--[[User:Z3126328|Jin Lee]] 16:06, 26 September 2009 (EST) congratulations to Group3! this is a really great assignment. overall, all the sections are well represented visually.
I liked the history part mostly! easy to read! Maybe the current research section can be improved by adding some images and relevant links. I found zebrafish is very interesting!thank you

--[[User:Z3187802|Vishnnu Shanmugam]] 19:32, 26 September 2009 (EST)Well done Zebra fish group. You've put together a well balanced assignment. The images under "Timeline and Stages of Embryonic Development" are impressive, really make the assignment stand out and the text flows nicely. I also noticed the links to researchers and research laboratories have been made throughout the text and because they are under specific headings, the reader will know what the information in the link will focus on. Additions to improve the assignment:

- there is some irrelevant images in the the text; the images "A 1981 issue of Nature journal" & "Development.jpg" are Unnecessary and could be replaced with others that support the text or summarize complex processes. (eg. motor neuron development in zebrafish or a time line showing the evolution in the use of zebrafish). The second image "Development.jpg" is better replaced with the image "A Zebrafish Pigment Mutant" as it is relevant to the text. The current research does not mension or explain the Zebrafish Pigment Mutant and thus is Unnecessary there.

- The Genetics section could do with an image of zebrafish chromosomes. This can be a simple hand drawn diagram and can be compared to the human chromosomal makeup.

- The current research section could use a description of how zebrafish research has impacted human embryology.

-The assignment needs to be properly referenced. see www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

- A Glossary would also complement the text.

Overall very impressive, only needs minor editing.

-[[User:Z3252340|Emily Wong]] 11:04, 27 September 2009 (EST) To begin with, you have done a great job group 3. It is a well researched, structured and organized page. The student contribution to the work is fairly even. The condensation of the written content is done well, with the combining of the timeline and stages in one section and extensive use of visual representation. However, information is not provided when clicking on the pictures as to what is in them. A description or explanation of what is occurring in each stage or at each time point would improve this project. Some of the images used have not been referenced properly in the image pages. Referencing is good and an extensive reference list has been provided. The content provided is very detailed by still brief and relevant to each section. This project could also be improved by adding some examples of current research and also referencing information throughout the written content. Perhaps an inclusion of a Pubmed search link in the current research section would be a good idea so that viewers can see what other research has been done using the zebrafish.

--[[User:Z3126345|Gang Liu]] 16:17, 27 September 2009 (EST)This is a very informative, well constructed and concise wikipage. It has demonstrated consistency throughout the page. There are a number of major subheadings have been included history, timeline, development, genetics and current embryology research as well as hand drawing. In addition, it has demonstrated extended research of literatures. For example, by dividing the history section in a number of subheadings such as "in the beginning", "Charles Kimmel", "Hesitations", etc. This has indicates the in-depth understanding of zebrafish embryo model use. It allows the reader to appreciate story behind the experiment, as well as scientists' thinking process and thoughts. "...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work...".

The graphic delivery of timeline and stages are very self-explanatory and powerful. A picture explains a thousand words. It makes the section easy to follow, and interesting to read also.

However, this project can be improved by considering the following points.

*I found genetic and current embryology section were very lengthy and wordy, and lack of pictures. In addition, referencing in these two sections were inconsistent with previous sections;

*Lack of glossary list. Such as "oxidative phosphrylation", "N-ethyl-N-nitrosourea (ENU)", "Diploid" and "Haploid" need to provide meanings;

*Inconsistent referencing. Name of author need to keep in alphabetical order. Also articles and web based materials are mixed. The first five references were inconsistent with the rest in style;

Last few words. I enjoyed reading it, learned something out of it. Big thumb up for me.

--[[User:Z3217686|Thomas Dangerfield]] 13:54, 28 September 2009 (EST)Well done guys! Nice, visually stimulating assignment! Just a few points about possibly having a little more written info on the timeline, just to help explain what is in each image. Also some pictures of the chromosomes of the zebrafish would be a handy visual tool. This may just be a little picky, but i notice that you have a few heading issues:

- Beginning not Begining. (In the begining..)

- Genetics of the Zebrafish and Embryology, and the you have a 'Genetics and Embryology' a couple of headings down which seems a little unnecessary.

Just as a hint on being thorough, maybe a glossary could have been included, as well as formatting for your references. Still, a well presented assignment has been done here!

--[[User:Z3185685|Sumaiya Rahman]] 16:06, 28 September 2009 (EST) Hey guys. Well done on the assignment overall. It was interesting to read about the zebrafish. The page was well introduced. It is good that you broke up the information using subheadings. This makes the page a lot easier to read as it breaks up the big slabs of text. I think it has been mentioned, but there are a couple of spelling mistakes. Also the sentence “This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work” needs to be fixed up. This is only a minor error. The history section could look a little bit less messy if the pictures were placed on the same side. “The fish is a frog... is a chicken... is a mouse” picture is very cute! But maybe a little bit too big because we need to scroll across to see it (or is that just me?). You have some great references and links and a great timetable and stages section with good visuals. The genetics and current research sections have a lot of text, maybe finding a way of making these sections more visually appealing would help the readers. Also including a glossary would help. Overall nice work!

--[[User:Z3215682|Carly Mooney]] 11:43, 29 September 2009 (EST)
Impressive, it looks as though much time and effort went into making the page easy to read yet informative.A few suggestions:
*some of the images I thought weren't entirely relevant, e.g.A 1981 issue of Nature journal" & "Development.jpg". maybe even crop it so that it is just the covers photograph in the image, rather than the entire journal.
*Maybe decrease the size of the fish is a frog is a chicken is a mouse jpg
*Maybe move the genetics picture further down as it is very similar and close to the timeline and stages section
*I think a glossary may help
*The references to be referenced properly, and the articles should have the pubmed number with their reference
*a few grammatical errors that need to be fixed up
These are all minor thoughts, well done.

--[[User:Z3254857|Begum Sonmez]] 14:53, 30 September 2009 (EST)
Hey Group 3. Let me start off by saying that I found your project on the Zebrafish unique. Under the history section:
*There were some sentences that were unnessesary such as 'Streisinger was not the only scientist working on the embryologic development of the zebrafish.'

*It was different and iteresting to learn about the different views of Streisinger and Kimmel. I enjoyed that fact that the structure wasn't entirely date than author than finding, but the inclussion of 'disputes and uncertainties' ('...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with.') as well.

*The information flows from Streisinger to Kimmel to hesitations.

*There is too much text bunched up under the sub-headings 'Taking the plunge', 'The Big Screen', and 'The Importance of Mutations'. Do not delete any text because I found it very interesting, but I suggest the use of Sub-sub headings, or some dot points. Another example would be to have the sub-heading as 'The Big Screen (1993-1996)'. This gets rid of some text.

The only thing that I found lacking under the Timeline/Stages section was a description of the image. I don't think a detailed descritption is necessary because the table is very well constructed. For example, state when the heart begins to develop or when it has finished developing, and place this information on the related image's page.

One of my favourite parts was the table under the Genetics section 'Mutations in Zebrafish Causing Developmental Heart Defects ' because the information is well constructed into a table format in a way that makes sense. In other words, the information is very clear. The information under 'How are mutations achieved?' was very interesting, I never before exactly thought about different ways of mutating.

I found a little problem in the first paragraph of current research section: 'Although the embryo was '''consistently''' studied '''continuously''' over the years...'. Just remove either bolded word. Some things that I think might enhance this section would be to include:
*Some links to published articles dealing with recent experimental research.
*Dates to identify when the research by The Laboratory of Molecular Genetics and Developmental Biology (China) was undertaken.
*The names of people/labortaories involved on the experiments on the gene of lissencephaly LIS1. In other words, inlude a specific example of a certain group of researchers that have utilised this gene.

And finally, the video under 'eye disorders' was interesting, relevant and informative at the same time. I liked it very much. Good work Group 3.

----

--[[User:S8600021|Mark Hill]] 01:44, 8 September 2009 (EST) A single image of a fish, well that really gets across the message of zebrafish development, interpret the information you have read and put it into a format that will interest the reader.

ZebraFish - Zebrafish are really cool...

I'm happy with zebrafish... never heard of them but they sound interesting :) what does everyone else think? ...Gaby Pinget

Great, who else is in our group then... Oh and i'm Sal by the way

Ok so i'm not sure who the other two are because i don't know your number but just for reference my number is z3218657.
I found this cool website with some pictures of Zebrafish embryo's developing... its pretty cool...

http://www.cas.vanderbilt.edu/bioimages/animals/danrer/zfish-devel.htm

and this website shows all the stages and times and such good for a timeline

http://zfin.org/zf_info/zfbook/stages/stages.html

Research Topics for this week:
- Timeline and Staging (Sal)
- History of Model Use (Gaby)
- Genetics (Bronwyn)
- Current Embryology Research (Jo)
--> Share what we've learnt with the group next session.

Hey Guys I have a lot of info... and i don't think i should post it all up here because there is a lot of stuff... Mainly all about the Staging though. I am going to put up a summary of the different stages. The website i gave you before ZFIN is the zebrafish database of model organisms. its great!

'''Zygote Period''' – Lasts for 0-0.75hours
Transition of one cell to two cells.
Fertilisation occurs activating cytoplastimic movement. The animal poles within the cell segregate the blastodisc from the yolk cytoplasm. Segregation continues into the cleavage staging.

'''Cleavage Period''' – Lasts for 0.75-2.25 hours
Transition from two cells to 128 cells
After the first cleavage the blastocysts division is approx every 15 minutes.

'''Blastula Period''' – Lasts for 2.25-5.25 hours
Transition from 128 cells to 50% epibolby
“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – Direct quote from the ZFIN website

'''Gastrula Period''' – Lasts for 5.25-10.33 hours
Transition from 50% epibolby to 1-4 somites
The gastrula period ends when epiboly is complete, and the tail bud has formed. Here each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations.

'''Segmentation period''' – Lasts from 10.33 – 24 hours
Transistion from 1-4 somites to Prim-5
Here dermis, vertebrae and skeletal muscle are formed

'''Pharyngula Period''' – Lasts from 24 – 48 hours
Transition from Prim 5 to Long-pec
The body axis begins to straighten and the fins begin to develop.

http://www.youtube.com/watch?v=0hGT667ktTw

'''Hatching Peroid''' – Last from 48 to 72hours
Transitions from Long-Pec to Protruding-mouth
In this period, primary organ systems develop and cartilage development begins.

'''Larval Period''' – Lasts from 72hours to 30days
transition from Protruding-mouth to Day 30-44
The pectoral fin continues to develop and the internal organs become more complex. Development continues.

'''Juvenille Period''' – Lasts from 30-44 days
Here adult fins and pigments as well as 12 teeth develop.

'''AdultPeriod''' – Lasts90days to 2 years
Full Breeding Adult.

http://www.youtube.com/watch?v=5ygcu9BRXI0 - Zebrafish heart beating!

http://www.youtube.com/watch?v=TbErcmhzUSY - alcohol effects on Zebrafish embryo

Websites used
http://dev.biologists.org/cgi/content/abstract/dev.022673v1

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

Hey! This is a really good site with a bunch of links relating to the different areas we have to research:
http://www.sanger.ac.uk/modelorgs/zebrafish.shtml

Hey guys.... Ive found a really good picture of the zebrafish embryo development (like the human one) but I dont know how to put it up, was thinking it might be better if it was actually on the page rather than a link to get the picture. JO

well I think that I just uploaded a picture but I have no idea where to... wow I'm so lost... Maybe that's a good thing because I have a feeling that it was copy right protected... DAMN COPY RIGHT ah ha ha

Hey all, all the websites that i have put up on this page have really good images... I have emailed the three of them and asked if it was ok if i used them to put up on here and i am awaiting a reply so
hopefully they will let us use them. Also i am having a tonne and a half of trouble uploading a InDesign Timeline image i created of the timeline and also having some issues with the net so i might
bring them to class and see if the computers there are readable other wise i am screwed and will just have to start all over again. I have a more complete Timeline and Staging format that i have
created as well. I'll put that up when i try and upload the image again. Peace. - Sal

Hey Hey So I got permission from Judy Cebra-Thomas to use all her images on her website which are each stage of development - YAY we just have to accredit them. So today apart from working on
unwinding my InDesign TImeline and putting the info up in bullets i am going to start to figure out whats happening in all the images and try an upload them onto the site so hopefully tomorrow we
will have an almost finished timeline and staging section! - Sal

Right so I've posted a link to the current sequencing status on the genetics part of our site. This updates pretty much everyday so I will be able to update ours right before submission so we have up-to-date info. This is however a minor detail so I'm writing it here to help us all remember! :) thanks

'''Hey guys!! please read this: I put up pictures which are really just to make it look pretty rather than add any info. Can you please tell me if you think they're stupid or not serious enough? I promise that I wont be offended! I understand if any of you think that we should take our assignment more seriously than that... it's just hard to find images for a history of zebrafish use!'''

I dont really get the light bulb lol. Sally were u able to get the embryo stage images as a link to the info as u wanted???? I think its a really good idea if we could do it, i think it might be a matter of asking someone who's actually good with computers to do it lol. Im still complying info for the current research section, currently in qld but i wanna have it up hopefully by this week, at least part of it anyways. Im gonna try n get some pictures but every place that ive asked think im an actual doctor lol so its been a bit hard.

Ok so not quite sure how to do the linking but i might just make it all link off the page into separate little pages... does that make sense??? Probably not but i can try explain it better on thursday. The
images take me 1.5hours each to upload because i have to make the image in indesign, Grab it and then transform the image to a PNG file as that is one that this website allows for uploading! So they
are coming up but ever so slowly! When i got permission to use the images i just told them I was a student studying Advance Science from UNSW. I was enrolled in an embryology course there where
we were doing an assignment on the Embryology of Zebrafish. Could i please use your images with referencing and a link to your webpage or document. Thats kind of what i said! i'm hoping to have it
all up and ready on thursday but might have to finish it friday night as have friends down from QLD that tend to make my place messy and have a house inspection on that ARVO!!! - Cheers SAL

Hey Sally, just so u know. I moved the websites you used to the references section so it didnt seem odd to keep it after ur info.

Hey Sally, we were thinkin about whether or not we could mix the timeline with the stages, so we thought that maybe if on the actual page we had the timeline with the pictures, then by clicking on the pictures there would be the info that u got or maybe even putting it all into a table so the pictures & info are more integrated.

Hey guys sorry i wasn't in class yesterday i had a funeral. What i was thinking of doing was actually moving all the data off the page and you click on it to go to another page which as all the info! but
the idea of clicking on the picture and going to it sounds great. One flaw is that is it ok if i use the pictures twice because i have been cutting, altering, adding text and flattening the images so that
they have a caption of what is happening. If i just cut another image to show each stage and then you click on that to go to it i think that could be cool as long as when you open the next page it still
has the information and the image with the caption of what is occurring! Hope thats ok. I will work on it all tonight and hopefully finish. Its taking a lot more time than expect sorry!Sal

hey Guys so I don't know if you guys have checked the website out yet but i have been working on it today! I continued what i was doing with the linked page and have also drawn my own diagrams
which are colour coded and easy for people to understand what is going on during the stages. I thought it looked cool. I haven't finished yet - a few more periods to go but i can't look at the screen
any more so i will do some on tuesday night! Hope thats ok. Just wondering though about the Nature article images above it kind of comes across the screen and i was wondering if we could somehow
move the image a little higher or enter down some spaces so that the timelines section is lower and the nature article doesn't come over the images pushing the table to the left! Let me know what you
all think. Sal

Hey Sal, your part of the project looks great! I really like the idea of clicking onto another page to get the info. works really really well and it's really easy to understand. As for the size of the picture obscuring your section, I think that you already thumbnailed it and it looks fine. Also, do you know how to put a youtube video onto the webpage? I was able to for a project in another class and it looked good but that was much easier because it had a link to a bunch of plug ins i could use. If anyone has any ideas please let me know! Gaby

Hey Sally, ur part looks awesome!!! Everyone done forget about your references...ive tried making the ones there so far look like it was done by one person lol. I dont know how to do the youtube clip thing but i have one too, was thinking maybe we should have a section for helpful links or do u think we should just put the links in with watever section its assiciated with. Wat do u guys think??? Jo

Hmm I was just thinking that the best thing to do was to put up the links strait after whatever they're associated with, that way they are supplimentary to what the reader is looking at rather than random extras at the end. That'll prob work best, right? Gaby

Thanks guys I've put a tonne of effort in so hope it pays off! I was going to fix up the references (cause they just have my websites without being real references) but it took me a lot longer than i thought for the images as you
can see its 5am. Nice. Yes i think linking the videos with the relevant parts is best. I have added links to videos throughout my section. Also I have made it so that when you put your mouse over the images it comes up with its own
reference/copy right info. Also when adding a link to another website its best to name the website instead of having just the web address. Its really easy if you don't know how and i can show you how to fix them up. When we get our
feedback from the other groups we can add and so forth before the project is actually marked which is really great. I think we need more info with the Genetics part but i figure bron is doing that tomorrow! Jo I really love your
sections pics and info it great and to the point!Gaby, yeah i did move it hope thats ok. I do have one question though and not meaning to hurt your feelings but i'm not that keen on the drawn image of the bird,fish,frog and mouse.
It doesn't really fit. Sorry, I mean obviously the decision is up to you, but i think it just looks a bit odd in the middle of the page because it you see it first and think , what? That was just some of the other feedback i got
from some of my friends that i asked to check out the website and see how they could maneuver around it. So that's my major spiel. Catch ya tomorrow. Sal

2009 Group Project 3

2009-10-14T23:07:31Z

Z3218657: /* Genetics of the ZebraFish and Embryology */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Beginning... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]

===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

[[File:A fish is a frog is a chicken is a mouse.jpg|thumbnail|left]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combining these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the left half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years from 1980s to 2009. Although the embryo was consistently studied over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fight cancer, to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understanding about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. These studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were however maintained as plastic phenotypes, expressing specific cell types including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology (MIT), Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence MIT determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Genetic Mutations Associated with Sight===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contributed to maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research http://www.youtube.com/watch?v=ZItgyfuxsfM

===Significance of Notochord===
One of the most significant characteristics of the embryo is the notochord; a midline structure required for skeletal development of the vertebrae and is the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Brain and Developmental Deficiencies===
The zebrafish has been utilized to express the gene of lissencephaly LIS1; a severe disease associated to brain malformation such as Miller-Dieker syndrome. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development within mature tissue samples. The LIS1 gene was predominately evident within brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. It was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149. PMID: 15138510

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967. PMID: 11691861

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513. PMID: 12468254

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724. PMID: 12209146

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

Zebrafish - Hatching Period

2009-10-14T04:38:55Z

Z3218657:

The duration of this period is from 48 to 72hours

* Transitions from Long-Pec to Protruding-mouth

* In this period, primary organ systems develop and cartilage development begins

* Pectoral fins are now elongated with a flat blade but still developing

* Jaws and Gills are also developing along side the pectoral fins

* Olfactory placodes are now developed in the anterior of the eye

* Hair Cells have differentiated

* The mouth is wide open and protrudes anteriorly just beyond the eye

* The first visible bone in the zebrafish, the cleithrum, can be seen between the first two myotomes

* Sub-intestinal vein is also prominent ventrally to the gut tract formation

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Pharyngula Period

2009-10-14T04:38:10Z

Z3218657:

The duration of this period is from 24 to 48 hours

* Transition from Prim 5 to Long-pec

* The body axis begins to straighten and the head straightens out and lifts dorsally

* Notochord is well developed

* Formation of the Dorsal and Ventral Stripe

* Nervous system is hollow and expanding anteriorly

* The brain has developed into 5 distinct lobes

* Seven pharyngeal arch's develop rapidly during this stage

* Pectoral fins begin to develop

* The Circulatory system develops and the heart beats for the first time

* Blood begins to circulate through a closed circuit of channels

* Tactile sensitivity appears and uncoordinated movements occur

[http://www.youtube.com/watch?v=5ygcu9BRXI0 Video of a Zebrafish Embryo Heart Beating]

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Segmentation Period

2009-10-14T04:37:22Z

Z3218657:

The Duration of this period is from 10.33 to 24 hours

* Transition from 1-4 somites to Prim-5

* Here dermis, vertebrae and skeletal muscle are formed

* The beginning of the primary organs develop

* The Embryo elongates and the tail bud (at the caudal end) becomes more prominent

* The first body movement occurs

* The Neural cord and the Notochord continue formation

* The Kupffer vessel in the Tail can be seen at its base

[[File:Segmentation_drawing.png|The Segmentation Period. Drawn and designed by Sally Clarke based off referenced material]]

Here you can see the development of the Kupffer Vessel and the the formation of the subdivisions of the brain located at the animal pole. The straightening out of the posterior trunk also occurs. The lumps along the dorsal neural tube show the formation of the hindbrain rhombomeres; divided segments of the neural tube within the hindbrain.

[http://www.youtube.com/watch?v=ahJjLzyioWM&feature=PlayList&p=49C439FE88128802&playnext=1&playnext_from=PL&index=7 Great Video of a Zebrafish egg development over 24 hours ]

[http://www.youtube.com/watch?v=6PhnHYZ5-zg&NR=1 Annotated zebrafish development timelapse]

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Gastrula Period

2009-10-14T04:36:57Z

Z3218657:

The duration of this period is from 5.25 to 10.33 hours

* Transition from 50% epiboly to 1-4 somites

* Primary germ layers (also known as 'germ ring') are produced during this stage

* Each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations

* Involution produces the germ ring by folding the blastoderm back upon itself

* Shield Stage occurs forming the dorsal side of the embryo sprouting pericardial regions.

* The Precordial plate within the animal pole forms the head structures

* The gastrula period ends when epiboly is complete, and the tail bud has form

[[File:Gastrula_This_one.png|The Gastrula Period. Drawn and designed by Sally Clarke based off referenced material]]

Here, there is a large number of cell migrations occurring to make sure each germ layer (endoderm, mesoderm, ectoderm) is in the right place so that bodily organs and tissues can form in the correct location. The shield then forms marking the dorsal side of the embryo and forming the pericardial region ventrally from the head region to the tail region. The PreCordial plate here forms the head structures. Lastly the tail bud forms indicating the end of epiboly. Effectively the blastoderm has covered the entire yoke.

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Blastula Period

2009-10-14T04:36:27Z

Z3218657:

The duration of this period is from 2.25 to 5.25 hours

* Transition from 128 cells to 50% epiboly

* It is called the Blastula period as the blastodisc begins to look ball like here until the onset of Gastrulation

* The orientation of the cleavage plains is indeterminate and the cells do not sit in an organized manner any more

* Embryo enters Mid-blastula Transition (MBT) where the yolk layer forms (YSL) which occurs when the cells that are closest to the yolk fuse to it and the epiboly begins

“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the Yoke Syncytial Layer (YSL) and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – [http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Embryonic Development of the Zebrafish, Kimmel et al.]

[[File:Blastula_period_Drawn_image2.png|The Bastula Period. Drawn and designed by Sally Clarke based off referenced material]]

During the Blastula Period the embryo changes shape from spherical to oblong. The egg goes through mid-blastula transition (MBT), which is when zygotic gene transcription occurs and the yoke syncytial layer (YSL) is formed when the cells closest too it fuse together. The beginning of epiboly begins during this period. This type of cell movement is where all embryonic cells move so that the ectoderm, or outer layer is on the outside of the forming embryo; however note here that the ectoderm does not completely form until the [[Gastrula Period]].

[http://www.youtube.com/watch?v=JSMJ_47_5D4 "Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate" (New Scientist magazine)]

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Cleavage Period

2009-10-14T04:35:43Z

Z3218657:

The duration of this period is from 0.75 to 2.25 hours

* The transition during this period is from two cells to 64 cells

* These transitions occur Rapidly and Synchronically

* After the first cleavage the blastomeres division is approximately every 15 minutes; There are six cleavages during this period

* A vertical furrow forms but when it reaches the end of the blastodisc, it become horizontal, and partially cleaves the zygote cell from the yoke cell, This is known as meroblastic divisions

* Thus the cells remain interconnected by cytoplasmic ‘bridges’.

[[File:Cleavage_internal.png|The Cleavage Period. Drawn and designed by Sally Clarke based off referenced material]]

During this period the blastomeres, which are the dividing cells singularly known as blastodisc, divide equally about every 15 minutes; As shown above.

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Zygote Period

2009-10-14T04:34:58Z

Z3218657:

The duration of this period is from 0 to 0.75 hours

* It is here that there is a transition from one cell to two cells

* Fertilisation occurs activating cytoplastimic movement

* The animal poles within the cell segregate the blastodisc from the yolk cytoplasm

* Segregation continues into the [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period]

[[File:ZygoteA.png|The Zygote Period. Drawn and designed by Sally Clarke based off referenced material]]

Here cytoplasm moves toward the animal pole to form the blastodisc, which is where future cell divisions will occur. The blastodisc then divides into 2 cells and this division continues occurring well into the cleavage stages.

[http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Zygote Period

2009-10-14T04:34:22Z

Z3218657:

The duration of this period is from 0 to 0.75 hours

* It is here that there is a transition from one cell to two cells

* Fertilisation occurs activating cytoplastimic movement

* The animal poles within the cell segregate the blastodisc from the yolk cytoplasm

* Segregation continues into the [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period]

[[File:ZygoteA.png|The Zygote Period. Drawn and designed by Sally Clarke based off referenced material]]

Here cytoplasm moves toward the animal pole to form the blastodisc, which is where future cell divisions will occur. The blastodisc then divides into 2 cells and this division continues occurring well into the cleavage stages.

[http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period]] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Cleavage Period

2009-10-14T04:33:48Z

Z3218657:

The duration of this period is from 0.75 to 2.25 hours

* The transition during this period is from two cells to 64 cells

* These transitions occur Rapidly and Synchronically

* After the first cleavage the blastomeres division is approximately every 15 minutes; There are six cleavages during this period

* A vertical furrow forms but when it reaches the end of the blastodisc, it become horizontal, and partially cleaves the zygote cell from the yoke cell, This is known as meroblastic divisions

* Thus the cells remain interconnected by cytoplasmic ‘bridges’.

[[File:Cleavage_internal.png|The Cleavage Period. Drawn and designed by Sally Clarke based off referenced material]]

During this period the blastomeres, which are the dividing cells singularly known as blastodisc, divide equally about every 15 minutes; As shown above.

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Gastrula_Period Gastrula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period]] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Zebrafish - Gastrula Period

2009-10-14T04:33:00Z

Z3218657:

The duration of this period is from 5.25 to 10.33 hours

* Transition from 50% epiboly to 1-4 somites

* Primary germ layers (also known as 'germ ring') are produced during this stage

* Each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations

* Involution produces the germ ring by folding the blastoderm back upon itself

* Shield Stage occurs forming the dorsal side of the embryo sprouting pericardial regions.

* The Precordial plate within the animal pole forms the head structures

* The gastrula period ends when epiboly is complete, and the tail bud has form

[[File:Gastrula_This_one.png|The Gastrula Period. Drawn and designed by Sally Clarke based off referenced material]]

Here, there is a large number of cell migrations occurring to make sure each germ layer (endoderm, mesoderm, ectoderm) is in the right place so that bodily organs and tissues can form in the correct location. The shield then forms marking the dorsal side of the embryo and forming the pericardial region ventrally from the head region to the tail region. The PreCordial plate here forms the head structures. Lastly the tail bud forms indicating the end of epiboly. Effectively the blastoderm has covered the entire yoke.

[http://php.med.unsw.edu.au/embryology/index.php?title=Zygote_Period Zygote Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Cleavage_Period Cleavage Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Blastula_Period Blastula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Segmentation_Period Segmentation Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Pharyngula_Period Pharyngula Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Hatching_Period Hatching Period] / [http://php.med.unsw.edu.au/embryology/index.php?title=Larval_Period Larval Period]] / [http://php.med.unsw.edu.au/embryology/index.php?title=2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development Home Page ]

Talk:2009 Group Project 3

2009-10-14T04:29:44Z

Z3218657: /* Project Updates */

== Project Updates ==

Glossary (jo)

Referencing (sally)

Spell check/grammar (bronwyn)

Pictures - delete some, add relevant ones

research paper linkages - more maybe (everyone)

Format of overall page

Condensing text??? (possibly no condensing required)

====Log of Changes ====

First section has been proofed- Bronwyn

Glossary has been added- Jo P.S. Let me know if Ive missed some and I will add them

Have linked the current research and pictures with their respective Institutes and Departments- Jo

I've done the Referencing but could someone have a look and see if its ok. i made all the web pages links and movies into links - i think they would be better in the texts but i don't know where they should all go. if anyone has any ideas please move them! - Sal

GABY - would you be able to fix your images please - changing the nature articles (or cutting them so just the picture part is showing) and by making the bird, fish, frog images smaller! )

== Lab 10 ==

--[[User:Z3283499|Antonio Lee]] 10:55, 2 October 2009 (EST) Hi everyone, I will be working with you during the Lab10 Tutorial and here is the news link and PDF of the manuscript for your group exercise. I encourage you to read the paper before the tutorial. Also, please indicate next to the questions below (using either your initials or student number) which one of the four questions you wish to address.
:'''Group 3 :''' [http://www.myelomabeacon.com/news/2009/09/21/study-shows-mozobil-induces-mobilization-of-stem-cells-but-not-tumor-cells/ '''Study Shows Mozobil Induces Mobilization Of Stem Cells But Not Myeloma Tumor Cells'''] in The Myeloma Beacon Published online 21 September 2009 10:28 pm [[Media:ANAT2341_Lab10_2009_Group 3 Reading.pdf|Manuscript (PDF): Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization]]

:Question 1. What is the background to the existing problem / disease condition? 3220040
:Question 2. What approach / method did the research team take to tackle / improve the problem? 3218657
:Question 3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance? 3223194
:Question 4. What are the next steps in moving forward? What are the next or new hurdles to overcome? 3218792

complications can be stopping this proliferation of the cells and the spread of them. also, contamination can still occur by tumor cells.

Maybe this treatment can be applied to other stem cells other than bone in order to repair/ regenerate various tissue types

Another development may be using the proliferation of bone stem cells to use for treatment of other things than cancer

==Constructive Criticism of Coordinator==
--[[User:S8600021|Mark Hill]] 08:07, 8 October 2009 (EST) The following comments are general in nature in no specific order, as it would be inappropriate to suggest specific changes and then assess the final project. Comments will be added during this week and you still have one week before final submission.

* There is no list of changes made to your project on the basis of peer assessments.
* Background history is well covered.
* [[2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development|Timeline_and_Stages_of_Embryonic_Development]] [[:File:Mutant_embryo.png]]no source information for these images. [[:File:Development.jpg]] Copyright, are they able to be reproduced? I have made a major point about image sources.
* Images of development are always good (if you are allowed to use) but there is no description of developmental events?
* How does this model differ from others? What are the differences between fish and men?
* Referencing within your project seems inconsistent.
* Have you grasped why the zebrafish is a good model of vertebrate development?
* You need to provide a consistent "feel" as well, this can sometimes be difficult when several different people are editing a project. Overall the project still seems a little "word heavy" and has not organised the current research well.
* You need to accurately proof-read your text " the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm" makes no sense. It also suggests that you have not reviewed your work sufficiently.

==Constructive Criticism of Peers==

--[[User:Z3258567|Sando Rashed]] 09:06, 1 October 2009 (EST)hey good work with the page, lots of good pieces of information, i really like the timeline and stages of the embryonic development but you might want to add just a few words to each stage just to make the page suitable for all people to read (someone with no background will look at it and understand it), with your background info i like the idea but there isnt really a sentence structure, you might want to put it in dot form as that what is sounds like when reading it. you might want to add a glossary to the bottom of the page and add words that are unknown to people to make the reading more comprehensive. you might want to add some more photos to the current research just to make that part of the assignment a bit more appealing as there is a lot of information to grasp there. with your referencing, it is incorrent format you might want to go on the internet and look up harvard referencing (http://www.unisa.edu.au/ltu/students/study/referencing/harvard.pdf)but overall good job :D

--[[User:Z3218146|Julianna Lam]] 01:22, 1 October 2009 (EST)

- the history part has way too much information. it needs to be more concise.

-the images used for staging and timeline is awesome. set out very cleary and very easy to read and understand

- the mutation table is a very good idea although you can probably fix it up a bit so the information doesnt look as crammed up.

- great current research!

--[[User:Z3252231|Angama Yaquobi]] 20:48, 30 September 2009 (EST)

Congrats everyone, your team has done a great job. The project looks amazing.
*The info in the introduction is very informative especially under the heading of "Why Zebrafish".
*The images used in the timeline and stages of embryonic development is awesome and clicking on the period name gives alot of great information. It also makes the webpage look very nice and tidy- its structed very well.
*The section for genetics was very well informative especially comparing the zebrafish genome with human genome.
*I would recommend to add more images related to each section to make the webpage look more visually eye-catchy.
Overall it was a great effort by all the team members.well done.

--[[User:Z3186093|Jenny Guy]] 18:30, 30 September 2009 (EST)

Improvements:
* Some of your sentences sound as though you've cut and pasted them from websites. For example, do you guys really used "combing" in your vocabulary.
* The history goes more into finding out about the scientists instead of what they fully achieved. Try focus on that instead of the scientists preferences etc.
* Throughout the entire page there isn't much referencing at all! You'll have many paragraphs which dont refer to any article/text. You either need to include referencing in your text or at least place an number in reference to where you found out that information (corresponding with your bibliography).
* You need to reference your "websites" in the bibliography. By typing the url you are not referencing. Most of the articles are referenced so make sure you use the same system with the websites.
* You used youtube??? oh dear. I can understand you using it to gain an understanding but that cannot be used as a reference. seriously, anyone can upload it. So instead find out WHO uploaded it and find out if they published it on a RELIABLE website. Copyright?
* One of the outcomes Mark has asked for is a list of "links to related resources/research laboratories?" Where is this?

--[[User:Z3255007|Sadaf Masood]] 15:29, 30 September 2009 (EST)Hey Group 3! Congrats on your project guys, well done!
Ill just list down few things that i liked and what could be done better:

1. Needs glossary guys, so some of the scientific jargon can be understood easily

2. Your timeline is excellent, it has great pictures, maybe a little description along with it might make it more clear.

3. The project only has four sections, i m sure we need to do a bit about fertilization and detalied embryonic steps as well

4. References to specific articles are provided which is very informative if anyone wants to do an in-depth analysis.

5. Formatting is impressive and page layout is great!

overall, this is an awesome project which is simple and straight-forward. Great work guys! And Best of Luck!!!

--[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

Group 3 well done impression effort , resulting in a great formatted page, which summarises the requirements of the outcomes needed, it was straight to the point and briefly covered the main topic in regards to the zebrafish.

- The assignment has come together extremely well only a few minor edit here and there needed.

- The illustrations regarding (Timeline and Stages of Embryonic Development) are great, it extremely enhances the wiki page assignment allowing the readers to visualize the information which stand out in regard to the information it come along great.
The links to specfic research laboratories have been made throughout the information giving the reader additional information to learn more on the topic which is a great reference.

Suggestions to improve the assignment:

1. illustrations such as :"Development.jpg" are should be replace which more appropriate images which are more relevant with the topic and the illustrations should be more regarded in the information for the reader to relate and understand visually.

2. The Genetics information should include specfic genetic and chromosomal imagery revealing the structure of chromosome in comparison to humans and should identify why they are different.

3. Current research information should reveal a brief explanation on how zebrafish research has impacted human embryology and how it used and why it used to improve or help develop research programs.

4. Referencing is a problem, specfic referencing is needed as the university has guidelines. Visit this webpage I think it will help: www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

5. Glossary will also help the readers to understand specfic terms and make the assignment flow better.

Impressive wiki page, minor editing should be undertaken but overall the outcome were covered. --[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

--[[User:Z3217015|Mitchell Mathieson]] 09:42, 25 September 2009 (EST)Overall very very nice. Was nice and succinct, and easy to read. The information was relevant, and the current research interesting. Maybe the stages on different pages was a bit difficult to read; I would have liked to have seen at least a bit of information on the main page, which expands to more on your secondary page. The formatting of the references I think needs to be looked at maybe.

--[[User:Z3224449|Elide Newton]] 14:38, 26 September 2009 (EST) HELLO GROUP 3: Congratulations on a great assignment. Each section is equally proportioned, and well represented visually. My one point of constructive criticism would be to the section on current research. Your information is great, as it shows how the zebrafish is being used as a model for different areas of research. The one thing which would improve this would be to include the dates of these research papers, just so the reader can know how recent the work is. maybe introduce the research as Person et al (2009) has used the zebrafish to.... Also are there any pictures on this research? Just because research language is so technical, some pictures would break it up. Hope this helps you group 3.

--[[User:Z3126328|Jin Lee]] 16:06, 26 September 2009 (EST) congratulations to Group3! this is a really great assignment. overall, all the sections are well represented visually.
I liked the history part mostly! easy to read! Maybe the current research section can be improved by adding some images and relevant links. I found zebrafish is very interesting!thank you

--[[User:Z3187802|Vishnnu Shanmugam]] 19:32, 26 September 2009 (EST)Well done Zebra fish group. You've put together a well balanced assignment. The images under "Timeline and Stages of Embryonic Development" are impressive, really make the assignment stand out and the text flows nicely. I also noticed the links to researchers and research laboratories have been made throughout the text and because they are under specific headings, the reader will know what the information in the link will focus on. Additions to improve the assignment:

- there is some irrelevant images in the the text; the images "A 1981 issue of Nature journal" & "Development.jpg" are Unnecessary and could be replaced with others that support the text or summarize complex processes. (eg. motor neuron development in zebrafish or a time line showing the evolution in the use of zebrafish). The second image "Development.jpg" is better replaced with the image "A Zebrafish Pigment Mutant" as it is relevant to the text. The current research does not mension or explain the Zebrafish Pigment Mutant and thus is Unnecessary there.

- The Genetics section could do with an image of zebrafish chromosomes. This can be a simple hand drawn diagram and can be compared to the human chromosomal makeup.

- The current research section could use a description of how zebrafish research has impacted human embryology.

-The assignment needs to be properly referenced. see www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

- A Glossary would also complement the text.

Overall very impressive, only needs minor editing.

-[[User:Z3252340|Emily Wong]] 11:04, 27 September 2009 (EST) To begin with, you have done a great job group 3. It is a well researched, structured and organized page. The student contribution to the work is fairly even. The condensation of the written content is done well, with the combining of the timeline and stages in one section and extensive use of visual representation. However, information is not provided when clicking on the pictures as to what is in them. A description or explanation of what is occurring in each stage or at each time point would improve this project. Some of the images used have not been referenced properly in the image pages. Referencing is good and an extensive reference list has been provided. The content provided is very detailed by still brief and relevant to each section. This project could also be improved by adding some examples of current research and also referencing information throughout the written content. Perhaps an inclusion of a Pubmed search link in the current research section would be a good idea so that viewers can see what other research has been done using the zebrafish.

--[[User:Z3126345|Gang Liu]] 16:17, 27 September 2009 (EST)This is a very informative, well constructed and concise wikipage. It has demonstrated consistency throughout the page. There are a number of major subheadings have been included history, timeline, development, genetics and current embryology research as well as hand drawing. In addition, it has demonstrated extended research of literatures. For example, by dividing the history section in a number of subheadings such as "in the beginning", "Charles Kimmel", "Hesitations", etc. This has indicates the in-depth understanding of zebrafish embryo model use. It allows the reader to appreciate story behind the experiment, as well as scientists' thinking process and thoughts. "...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work...".

The graphic delivery of timeline and stages are very self-explanatory and powerful. A picture explains a thousand words. It makes the section easy to follow, and interesting to read also.

However, this project can be improved by considering the following points.

*I found genetic and current embryology section were very lengthy and wordy, and lack of pictures. In addition, referencing in these two sections were inconsistent with previous sections;

*Lack of glossary list. Such as "oxidative phosphrylation", "N-ethyl-N-nitrosourea (ENU)", "Diploid" and "Haploid" need to provide meanings;

*Inconsistent referencing. Name of author need to keep in alphabetical order. Also articles and web based materials are mixed. The first five references were inconsistent with the rest in style;

Last few words. I enjoyed reading it, learned something out of it. Big thumb up for me.

--[[User:Z3217686|Thomas Dangerfield]] 13:54, 28 September 2009 (EST)Well done guys! Nice, visually stimulating assignment! Just a few points about possibly having a little more written info on the timeline, just to help explain what is in each image. Also some pictures of the chromosomes of the zebrafish would be a handy visual tool. This may just be a little picky, but i notice that you have a few heading issues:

- Beginning not Begining. (In the begining..)

- Genetics of the Zebrafish and Embryology, and the you have a 'Genetics and Embryology' a couple of headings down which seems a little unnecessary.

Just as a hint on being thorough, maybe a glossary could have been included, as well as formatting for your references. Still, a well presented assignment has been done here!

--[[User:Z3185685|Sumaiya Rahman]] 16:06, 28 September 2009 (EST) Hey guys. Well done on the assignment overall. It was interesting to read about the zebrafish. The page was well introduced. It is good that you broke up the information using subheadings. This makes the page a lot easier to read as it breaks up the big slabs of text. I think it has been mentioned, but there are a couple of spelling mistakes. Also the sentence “This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work” needs to be fixed up. This is only a minor error. The history section could look a little bit less messy if the pictures were placed on the same side. “The fish is a frog... is a chicken... is a mouse” picture is very cute! But maybe a little bit too big because we need to scroll across to see it (or is that just me?). You have some great references and links and a great timetable and stages section with good visuals. The genetics and current research sections have a lot of text, maybe finding a way of making these sections more visually appealing would help the readers. Also including a glossary would help. Overall nice work!

--[[User:Z3215682|Carly Mooney]] 11:43, 29 September 2009 (EST)
Impressive, it looks as though much time and effort went into making the page easy to read yet informative.A few suggestions:
*some of the images I thought weren't entirely relevant, e.g.A 1981 issue of Nature journal" & "Development.jpg". maybe even crop it so that it is just the covers photograph in the image, rather than the entire journal.
*Maybe decrease the size of the fish is a frog is a chicken is a mouse jpg
*Maybe move the genetics picture further down as it is very similar and close to the timeline and stages section
*I think a glossary may help
*The references to be referenced properly, and the articles should have the pubmed number with their reference
*a few grammatical errors that need to be fixed up
These are all minor thoughts, well done.

--[[User:Z3254857|Begum Sonmez]] 14:53, 30 September 2009 (EST)
Hey Group 3. Let me start off by saying that I found your project on the Zebrafish unique. Under the history section:
*There were some sentences that were unnessesary such as 'Streisinger was not the only scientist working on the embryologic development of the zebrafish.'

*It was different and iteresting to learn about the different views of Streisinger and Kimmel. I enjoyed that fact that the structure wasn't entirely date than author than finding, but the inclussion of 'disputes and uncertainties' ('...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with.') as well.

*The information flows from Streisinger to Kimmel to hesitations.

*There is too much text bunched up under the sub-headings 'Taking the plunge', 'The Big Screen', and 'The Importance of Mutations'. Do not delete any text because I found it very interesting, but I suggest the use of Sub-sub headings, or some dot points. Another example would be to have the sub-heading as 'The Big Screen (1993-1996)'. This gets rid of some text.

The only thing that I found lacking under the Timeline/Stages section was a description of the image. I don't think a detailed descritption is necessary because the table is very well constructed. For example, state when the heart begins to develop or when it has finished developing, and place this information on the related image's page.

One of my favourite parts was the table under the Genetics section 'Mutations in Zebrafish Causing Developmental Heart Defects ' because the information is well constructed into a table format in a way that makes sense. In other words, the information is very clear. The information under 'How are mutations achieved?' was very interesting, I never before exactly thought about different ways of mutating.

I found a little problem in the first paragraph of current research section: 'Although the embryo was '''consistently''' studied '''continuously''' over the years...'. Just remove either bolded word. Some things that I think might enhance this section would be to include:
*Some links to published articles dealing with recent experimental research.
*Dates to identify when the research by The Laboratory of Molecular Genetics and Developmental Biology (China) was undertaken.
*The names of people/labortaories involved on the experiments on the gene of lissencephaly LIS1. In other words, inlude a specific example of a certain group of researchers that have utilised this gene.

And finally, the video under 'eye disorders' was interesting, relevant and informative at the same time. I liked it very much. Good work Group 3.

----

--[[User:S8600021|Mark Hill]] 01:44, 8 September 2009 (EST) A single image of a fish, well that really gets across the message of zebrafish development, interpret the information you have read and put it into a format that will interest the reader.

ZebraFish - Zebrafish are really cool...

I'm happy with zebrafish... never heard of them but they sound interesting :) what does everyone else think? ...Gaby Pinget

Great, who else is in our group then... Oh and i'm Sal by the way

Ok so i'm not sure who the other two are because i don't know your number but just for reference my number is z3218657.
I found this cool website with some pictures of Zebrafish embryo's developing... its pretty cool...

http://www.cas.vanderbilt.edu/bioimages/animals/danrer/zfish-devel.htm

and this website shows all the stages and times and such good for a timeline

http://zfin.org/zf_info/zfbook/stages/stages.html

Research Topics for this week:
- Timeline and Staging (Sal)
- History of Model Use (Gaby)
- Genetics (Bronwyn)
- Current Embryology Research (Jo)
--> Share what we've learnt with the group next session.

Hey Guys I have a lot of info... and i don't think i should post it all up here because there is a lot of stuff... Mainly all about the Staging though. I am going to put up a summary of the different stages. The website i gave you before ZFIN is the zebrafish database of model organisms. its great!

'''Zygote Period''' – Lasts for 0-0.75hours
Transition of one cell to two cells.
Fertilisation occurs activating cytoplastimic movement. The animal poles within the cell segregate the blastodisc from the yolk cytoplasm. Segregation continues into the cleavage staging.

'''Cleavage Period''' – Lasts for 0.75-2.25 hours
Transition from two cells to 128 cells
After the first cleavage the blastocysts division is approx every 15 minutes.

'''Blastula Period''' – Lasts for 2.25-5.25 hours
Transition from 128 cells to 50% epibolby
“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – Direct quote from the ZFIN website

'''Gastrula Period''' – Lasts for 5.25-10.33 hours
Transition from 50% epibolby to 1-4 somites
The gastrula period ends when epiboly is complete, and the tail bud has formed. Here each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations.

'''Segmentation period''' – Lasts from 10.33 – 24 hours
Transistion from 1-4 somites to Prim-5
Here dermis, vertebrae and skeletal muscle are formed

'''Pharyngula Period''' – Lasts from 24 – 48 hours
Transition from Prim 5 to Long-pec
The body axis begins to straighten and the fins begin to develop.

http://www.youtube.com/watch?v=0hGT667ktTw

'''Hatching Peroid''' – Last from 48 to 72hours
Transitions from Long-Pec to Protruding-mouth
In this period, primary organ systems develop and cartilage development begins.

'''Larval Period''' – Lasts from 72hours to 30days
transition from Protruding-mouth to Day 30-44
The pectoral fin continues to develop and the internal organs become more complex. Development continues.

'''Juvenille Period''' – Lasts from 30-44 days
Here adult fins and pigments as well as 12 teeth develop.

'''AdultPeriod''' – Lasts90days to 2 years
Full Breeding Adult.

http://www.youtube.com/watch?v=5ygcu9BRXI0 - Zebrafish heart beating!

http://www.youtube.com/watch?v=TbErcmhzUSY - alcohol effects on Zebrafish embryo

Websites used
http://dev.biologists.org/cgi/content/abstract/dev.022673v1

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

Hey! This is a really good site with a bunch of links relating to the different areas we have to research:
http://www.sanger.ac.uk/modelorgs/zebrafish.shtml

Hey guys.... Ive found a really good picture of the zebrafish embryo development (like the human one) but I dont know how to put it up, was thinking it might be better if it was actually on the page rather than a link to get the picture. JO

well I think that I just uploaded a picture but I have no idea where to... wow I'm so lost... Maybe that's a good thing because I have a feeling that it was copy right protected... DAMN COPY RIGHT ah ha ha

Hey all, all the websites that i have put up on this page have really good images... I have emailed the three of them and asked if it was ok if i used them to put up on here and i am awaiting a reply so
hopefully they will let us use them. Also i am having a tonne and a half of trouble uploading a InDesign Timeline image i created of the timeline and also having some issues with the net so i might
bring them to class and see if the computers there are readable other wise i am screwed and will just have to start all over again. I have a more complete Timeline and Staging format that i have
created as well. I'll put that up when i try and upload the image again. Peace. - Sal

Hey Hey So I got permission from Judy Cebra-Thomas to use all her images on her website which are each stage of development - YAY we just have to accredit them. So today apart from working on
unwinding my InDesign TImeline and putting the info up in bullets i am going to start to figure out whats happening in all the images and try an upload them onto the site so hopefully tomorrow we
will have an almost finished timeline and staging section! - Sal

Right so I've posted a link to the current sequencing status on the genetics part of our site. This updates pretty much everyday so I will be able to update ours right before submission so we have up-to-date info. This is however a minor detail so I'm writing it here to help us all remember! :) thanks

'''Hey guys!! please read this: I put up pictures which are really just to make it look pretty rather than add any info. Can you please tell me if you think they're stupid or not serious enough? I promise that I wont be offended! I understand if any of you think that we should take our assignment more seriously than that... it's just hard to find images for a history of zebrafish use!'''

I dont really get the light bulb lol. Sally were u able to get the embryo stage images as a link to the info as u wanted???? I think its a really good idea if we could do it, i think it might be a matter of asking someone who's actually good with computers to do it lol. Im still complying info for the current research section, currently in qld but i wanna have it up hopefully by this week, at least part of it anyways. Im gonna try n get some pictures but every place that ive asked think im an actual doctor lol so its been a bit hard.

Ok so not quite sure how to do the linking but i might just make it all link off the page into separate little pages... does that make sense??? Probably not but i can try explain it better on thursday. The
images take me 1.5hours each to upload because i have to make the image in indesign, Grab it and then transform the image to a PNG file as that is one that this website allows for uploading! So they
are coming up but ever so slowly! When i got permission to use the images i just told them I was a student studying Advance Science from UNSW. I was enrolled in an embryology course there where
we were doing an assignment on the Embryology of Zebrafish. Could i please use your images with referencing and a link to your webpage or document. Thats kind of what i said! i'm hoping to have it
all up and ready on thursday but might have to finish it friday night as have friends down from QLD that tend to make my place messy and have a house inspection on that ARVO!!! - Cheers SAL

Hey Sally, just so u know. I moved the websites you used to the references section so it didnt seem odd to keep it after ur info.

Hey Sally, we were thinkin about whether or not we could mix the timeline with the stages, so we thought that maybe if on the actual page we had the timeline with the pictures, then by clicking on the pictures there would be the info that u got or maybe even putting it all into a table so the pictures & info are more integrated.

Hey guys sorry i wasn't in class yesterday i had a funeral. What i was thinking of doing was actually moving all the data off the page and you click on it to go to another page which as all the info! but
the idea of clicking on the picture and going to it sounds great. One flaw is that is it ok if i use the pictures twice because i have been cutting, altering, adding text and flattening the images so that
they have a caption of what is happening. If i just cut another image to show each stage and then you click on that to go to it i think that could be cool as long as when you open the next page it still
has the information and the image with the caption of what is occurring! Hope thats ok. I will work on it all tonight and hopefully finish. Its taking a lot more time than expect sorry!Sal

hey Guys so I don't know if you guys have checked the website out yet but i have been working on it today! I continued what i was doing with the linked page and have also drawn my own diagrams
which are colour coded and easy for people to understand what is going on during the stages. I thought it looked cool. I haven't finished yet - a few more periods to go but i can't look at the screen
any more so i will do some on tuesday night! Hope thats ok. Just wondering though about the Nature article images above it kind of comes across the screen and i was wondering if we could somehow
move the image a little higher or enter down some spaces so that the timelines section is lower and the nature article doesn't come over the images pushing the table to the left! Let me know what you
all think. Sal

Hey Sal, your part of the project looks great! I really like the idea of clicking onto another page to get the info. works really really well and it's really easy to understand. As for the size of the picture obscuring your section, I think that you already thumbnailed it and it looks fine. Also, do you know how to put a youtube video onto the webpage? I was able to for a project in another class and it looked good but that was much easier because it had a link to a bunch of plug ins i could use. If anyone has any ideas please let me know! Gaby

Hey Sally, ur part looks awesome!!! Everyone done forget about your references...ive tried making the ones there so far look like it was done by one person lol. I dont know how to do the youtube clip thing but i have one too, was thinking maybe we should have a section for helpful links or do u think we should just put the links in with watever section its assiciated with. Wat do u guys think??? Jo

Hmm I was just thinking that the best thing to do was to put up the links strait after whatever they're associated with, that way they are supplimentary to what the reader is looking at rather than random extras at the end. That'll prob work best, right? Gaby

Thanks guys I've put a tonne of effort in so hope it pays off! I was going to fix up the references (cause they just have my websites without being real references) but it took me a lot longer than i thought for the images as you
can see its 5am. Nice. Yes i think linking the videos with the relevant parts is best. I have added links to videos throughout my section. Also I have made it so that when you put your mouse over the images it comes up with its own
reference/copy right info. Also when adding a link to another website its best to name the website instead of having just the web address. Its really easy if you don't know how and i can show you how to fix them up. When we get our
feedback from the other groups we can add and so forth before the project is actually marked which is really great. I think we need more info with the Genetics part but i figure bron is doing that tomorrow! Jo I really love your
sections pics and info it great and to the point!Gaby, yeah i did move it hope thats ok. I do have one question though and not meaning to hurt your feelings but i'm not that keen on the drawn image of the bird,fish,frog and mouse.
It doesn't really fit. Sorry, I mean obviously the decision is up to you, but i think it just looks a bit odd in the middle of the page because it you see it first and think , what? That was just some of the other feedback i got
from some of my friends that i asked to check out the website and see how they could maneuver around it. So that's my major spiel. Catch ya tomorrow. Sal

Talk:2009 Group Project 3

2009-10-14T04:27:29Z

Z3218657: /* Project Updates */

== Project Updates ==

Glossary (jo)

Referencing (sally)

Spell check/grammar (bronwyn)

Pictures - delete some, add relevant ones

research paper linkages - more maybe (everyone)

Format of overall page

Condensing text??? (possibly no condensing required)

====Log of Changes ====

First section has been proofed- Bronwyn

Glossary has been added- Jo P.S. Let me know if Ive missed some and I will add them

Have linked the current research and pictures with their respective Institutes and Departments- Jo

I've done the Referencing but could someone have a look and see if its ok. i made all the web pages links and movies into links - i think they would be better in the texts but i don't know where they should all go. if anyone has any ideas please move them! - Sal

== Lab 10 ==

--[[User:Z3283499|Antonio Lee]] 10:55, 2 October 2009 (EST) Hi everyone, I will be working with you during the Lab10 Tutorial and here is the news link and PDF of the manuscript for your group exercise. I encourage you to read the paper before the tutorial. Also, please indicate next to the questions below (using either your initials or student number) which one of the four questions you wish to address.
:'''Group 3 :''' [http://www.myelomabeacon.com/news/2009/09/21/study-shows-mozobil-induces-mobilization-of-stem-cells-but-not-tumor-cells/ '''Study Shows Mozobil Induces Mobilization Of Stem Cells But Not Myeloma Tumor Cells'''] in The Myeloma Beacon Published online 21 September 2009 10:28 pm [[Media:ANAT2341_Lab10_2009_Group 3 Reading.pdf|Manuscript (PDF): Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization]]

:Question 1. What is the background to the existing problem / disease condition? 3220040
:Question 2. What approach / method did the research team take to tackle / improve the problem? 3218657
:Question 3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance? 3223194
:Question 4. What are the next steps in moving forward? What are the next or new hurdles to overcome? 3218792

complications can be stopping this proliferation of the cells and the spread of them. also, contamination can still occur by tumor cells.

Maybe this treatment can be applied to other stem cells other than bone in order to repair/ regenerate various tissue types

Another development may be using the proliferation of bone stem cells to use for treatment of other things than cancer

==Constructive Criticism of Coordinator==
--[[User:S8600021|Mark Hill]] 08:07, 8 October 2009 (EST) The following comments are general in nature in no specific order, as it would be inappropriate to suggest specific changes and then assess the final project. Comments will be added during this week and you still have one week before final submission.

* There is no list of changes made to your project on the basis of peer assessments.
* Background history is well covered.
* [[2009_Group_Project_3#Timeline_and_Stages_of_Embryonic_Development|Timeline_and_Stages_of_Embryonic_Development]] [[:File:Mutant_embryo.png]]no source information for these images. [[:File:Development.jpg]] Copyright, are they able to be reproduced? I have made a major point about image sources.
* Images of development are always good (if you are allowed to use) but there is no description of developmental events?
* How does this model differ from others? What are the differences between fish and men?
* Referencing within your project seems inconsistent.
* Have you grasped why the zebrafish is a good model of vertebrate development?
* You need to provide a consistent "feel" as well, this can sometimes be difficult when several different people are editing a project. Overall the project still seems a little "word heavy" and has not organised the current research well.
* You need to accurately proof-read your text " the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm" makes no sense. It also suggests that you have not reviewed your work sufficiently.

==Constructive Criticism of Peers==

--[[User:Z3258567|Sando Rashed]] 09:06, 1 October 2009 (EST)hey good work with the page, lots of good pieces of information, i really like the timeline and stages of the embryonic development but you might want to add just a few words to each stage just to make the page suitable for all people to read (someone with no background will look at it and understand it), with your background info i like the idea but there isnt really a sentence structure, you might want to put it in dot form as that what is sounds like when reading it. you might want to add a glossary to the bottom of the page and add words that are unknown to people to make the reading more comprehensive. you might want to add some more photos to the current research just to make that part of the assignment a bit more appealing as there is a lot of information to grasp there. with your referencing, it is incorrent format you might want to go on the internet and look up harvard referencing (http://www.unisa.edu.au/ltu/students/study/referencing/harvard.pdf)but overall good job :D

--[[User:Z3218146|Julianna Lam]] 01:22, 1 October 2009 (EST)

- the history part has way too much information. it needs to be more concise.

-the images used for staging and timeline is awesome. set out very cleary and very easy to read and understand

- the mutation table is a very good idea although you can probably fix it up a bit so the information doesnt look as crammed up.

- great current research!

--[[User:Z3252231|Angama Yaquobi]] 20:48, 30 September 2009 (EST)

Congrats everyone, your team has done a great job. The project looks amazing.
*The info in the introduction is very informative especially under the heading of "Why Zebrafish".
*The images used in the timeline and stages of embryonic development is awesome and clicking on the period name gives alot of great information. It also makes the webpage look very nice and tidy- its structed very well.
*The section for genetics was very well informative especially comparing the zebrafish genome with human genome.
*I would recommend to add more images related to each section to make the webpage look more visually eye-catchy.
Overall it was a great effort by all the team members.well done.

--[[User:Z3186093|Jenny Guy]] 18:30, 30 September 2009 (EST)

Improvements:
* Some of your sentences sound as though you've cut and pasted them from websites. For example, do you guys really used "combing" in your vocabulary.
* The history goes more into finding out about the scientists instead of what they fully achieved. Try focus on that instead of the scientists preferences etc.
* Throughout the entire page there isn't much referencing at all! You'll have many paragraphs which dont refer to any article/text. You either need to include referencing in your text or at least place an number in reference to where you found out that information (corresponding with your bibliography).
* You need to reference your "websites" in the bibliography. By typing the url you are not referencing. Most of the articles are referenced so make sure you use the same system with the websites.
* You used youtube??? oh dear. I can understand you using it to gain an understanding but that cannot be used as a reference. seriously, anyone can upload it. So instead find out WHO uploaded it and find out if they published it on a RELIABLE website. Copyright?
* One of the outcomes Mark has asked for is a list of "links to related resources/research laboratories?" Where is this?

--[[User:Z3255007|Sadaf Masood]] 15:29, 30 September 2009 (EST)Hey Group 3! Congrats on your project guys, well done!
Ill just list down few things that i liked and what could be done better:

1. Needs glossary guys, so some of the scientific jargon can be understood easily

2. Your timeline is excellent, it has great pictures, maybe a little description along with it might make it more clear.

3. The project only has four sections, i m sure we need to do a bit about fertilization and detalied embryonic steps as well

4. References to specific articles are provided which is very informative if anyone wants to do an in-depth analysis.

5. Formatting is impressive and page layout is great!

overall, this is an awesome project which is simple and straight-forward. Great work guys! And Best of Luck!!!

--[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

Group 3 well done impression effort , resulting in a great formatted page, which summarises the requirements of the outcomes needed, it was straight to the point and briefly covered the main topic in regards to the zebrafish.

- The assignment has come together extremely well only a few minor edit here and there needed.

- The illustrations regarding (Timeline and Stages of Embryonic Development) are great, it extremely enhances the wiki page assignment allowing the readers to visualize the information which stand out in regard to the information it come along great.
The links to specfic research laboratories have been made throughout the information giving the reader additional information to learn more on the topic which is a great reference.

Suggestions to improve the assignment:

1. illustrations such as :"Development.jpg" are should be replace which more appropriate images which are more relevant with the topic and the illustrations should be more regarded in the information for the reader to relate and understand visually.

2. The Genetics information should include specfic genetic and chromosomal imagery revealing the structure of chromosome in comparison to humans and should identify why they are different.

3. Current research information should reveal a brief explanation on how zebrafish research has impacted human embryology and how it used and why it used to improve or help develop research programs.

4. Referencing is a problem, specfic referencing is needed as the university has guidelines. Visit this webpage I think it will help: www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

5. Glossary will also help the readers to understand specfic terms and make the assignment flow better.

Impressive wiki page, minor editing should be undertaken but overall the outcome were covered. --[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

--[[User:Z3217015|Mitchell Mathieson]] 09:42, 25 September 2009 (EST)Overall very very nice. Was nice and succinct, and easy to read. The information was relevant, and the current research interesting. Maybe the stages on different pages was a bit difficult to read; I would have liked to have seen at least a bit of information on the main page, which expands to more on your secondary page. The formatting of the references I think needs to be looked at maybe.

--[[User:Z3224449|Elide Newton]] 14:38, 26 September 2009 (EST) HELLO GROUP 3: Congratulations on a great assignment. Each section is equally proportioned, and well represented visually. My one point of constructive criticism would be to the section on current research. Your information is great, as it shows how the zebrafish is being used as a model for different areas of research. The one thing which would improve this would be to include the dates of these research papers, just so the reader can know how recent the work is. maybe introduce the research as Person et al (2009) has used the zebrafish to.... Also are there any pictures on this research? Just because research language is so technical, some pictures would break it up. Hope this helps you group 3.

--[[User:Z3126328|Jin Lee]] 16:06, 26 September 2009 (EST) congratulations to Group3! this is a really great assignment. overall, all the sections are well represented visually.
I liked the history part mostly! easy to read! Maybe the current research section can be improved by adding some images and relevant links. I found zebrafish is very interesting!thank you

--[[User:Z3187802|Vishnnu Shanmugam]] 19:32, 26 September 2009 (EST)Well done Zebra fish group. You've put together a well balanced assignment. The images under "Timeline and Stages of Embryonic Development" are impressive, really make the assignment stand out and the text flows nicely. I also noticed the links to researchers and research laboratories have been made throughout the text and because they are under specific headings, the reader will know what the information in the link will focus on. Additions to improve the assignment:

- there is some irrelevant images in the the text; the images "A 1981 issue of Nature journal" & "Development.jpg" are Unnecessary and could be replaced with others that support the text or summarize complex processes. (eg. motor neuron development in zebrafish or a time line showing the evolution in the use of zebrafish). The second image "Development.jpg" is better replaced with the image "A Zebrafish Pigment Mutant" as it is relevant to the text. The current research does not mension or explain the Zebrafish Pigment Mutant and thus is Unnecessary there.

- The Genetics section could do with an image of zebrafish chromosomes. This can be a simple hand drawn diagram and can be compared to the human chromosomal makeup.

- The current research section could use a description of how zebrafish research has impacted human embryology.

-The assignment needs to be properly referenced. see www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

- A Glossary would also complement the text.

Overall very impressive, only needs minor editing.

-[[User:Z3252340|Emily Wong]] 11:04, 27 September 2009 (EST) To begin with, you have done a great job group 3. It is a well researched, structured and organized page. The student contribution to the work is fairly even. The condensation of the written content is done well, with the combining of the timeline and stages in one section and extensive use of visual representation. However, information is not provided when clicking on the pictures as to what is in them. A description or explanation of what is occurring in each stage or at each time point would improve this project. Some of the images used have not been referenced properly in the image pages. Referencing is good and an extensive reference list has been provided. The content provided is very detailed by still brief and relevant to each section. This project could also be improved by adding some examples of current research and also referencing information throughout the written content. Perhaps an inclusion of a Pubmed search link in the current research section would be a good idea so that viewers can see what other research has been done using the zebrafish.

--[[User:Z3126345|Gang Liu]] 16:17, 27 September 2009 (EST)This is a very informative, well constructed and concise wikipage. It has demonstrated consistency throughout the page. There are a number of major subheadings have been included history, timeline, development, genetics and current embryology research as well as hand drawing. In addition, it has demonstrated extended research of literatures. For example, by dividing the history section in a number of subheadings such as "in the beginning", "Charles Kimmel", "Hesitations", etc. This has indicates the in-depth understanding of zebrafish embryo model use. It allows the reader to appreciate story behind the experiment, as well as scientists' thinking process and thoughts. "...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work...".

The graphic delivery of timeline and stages are very self-explanatory and powerful. A picture explains a thousand words. It makes the section easy to follow, and interesting to read also.

However, this project can be improved by considering the following points.

*I found genetic and current embryology section were very lengthy and wordy, and lack of pictures. In addition, referencing in these two sections were inconsistent with previous sections;

*Lack of glossary list. Such as "oxidative phosphrylation", "N-ethyl-N-nitrosourea (ENU)", "Diploid" and "Haploid" need to provide meanings;

*Inconsistent referencing. Name of author need to keep in alphabetical order. Also articles and web based materials are mixed. The first five references were inconsistent with the rest in style;

Last few words. I enjoyed reading it, learned something out of it. Big thumb up for me.

--[[User:Z3217686|Thomas Dangerfield]] 13:54, 28 September 2009 (EST)Well done guys! Nice, visually stimulating assignment! Just a few points about possibly having a little more written info on the timeline, just to help explain what is in each image. Also some pictures of the chromosomes of the zebrafish would be a handy visual tool. This may just be a little picky, but i notice that you have a few heading issues:

- Beginning not Begining. (In the begining..)

- Genetics of the Zebrafish and Embryology, and the you have a 'Genetics and Embryology' a couple of headings down which seems a little unnecessary.

Just as a hint on being thorough, maybe a glossary could have been included, as well as formatting for your references. Still, a well presented assignment has been done here!

--[[User:Z3185685|Sumaiya Rahman]] 16:06, 28 September 2009 (EST) Hey guys. Well done on the assignment overall. It was interesting to read about the zebrafish. The page was well introduced. It is good that you broke up the information using subheadings. This makes the page a lot easier to read as it breaks up the big slabs of text. I think it has been mentioned, but there are a couple of spelling mistakes. Also the sentence “This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work” needs to be fixed up. This is only a minor error. The history section could look a little bit less messy if the pictures were placed on the same side. “The fish is a frog... is a chicken... is a mouse” picture is very cute! But maybe a little bit too big because we need to scroll across to see it (or is that just me?). You have some great references and links and a great timetable and stages section with good visuals. The genetics and current research sections have a lot of text, maybe finding a way of making these sections more visually appealing would help the readers. Also including a glossary would help. Overall nice work!

--[[User:Z3215682|Carly Mooney]] 11:43, 29 September 2009 (EST)
Impressive, it looks as though much time and effort went into making the page easy to read yet informative.A few suggestions:
*some of the images I thought weren't entirely relevant, e.g.A 1981 issue of Nature journal" & "Development.jpg". maybe even crop it so that it is just the covers photograph in the image, rather than the entire journal.
*Maybe decrease the size of the fish is a frog is a chicken is a mouse jpg
*Maybe move the genetics picture further down as it is very similar and close to the timeline and stages section
*I think a glossary may help
*The references to be referenced properly, and the articles should have the pubmed number with their reference
*a few grammatical errors that need to be fixed up
These are all minor thoughts, well done.

--[[User:Z3254857|Begum Sonmez]] 14:53, 30 September 2009 (EST)
Hey Group 3. Let me start off by saying that I found your project on the Zebrafish unique. Under the history section:
*There were some sentences that were unnessesary such as 'Streisinger was not the only scientist working on the embryologic development of the zebrafish.'

*It was different and iteresting to learn about the different views of Streisinger and Kimmel. I enjoyed that fact that the structure wasn't entirely date than author than finding, but the inclussion of 'disputes and uncertainties' ('...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with.') as well.

*The information flows from Streisinger to Kimmel to hesitations.

*There is too much text bunched up under the sub-headings 'Taking the plunge', 'The Big Screen', and 'The Importance of Mutations'. Do not delete any text because I found it very interesting, but I suggest the use of Sub-sub headings, or some dot points. Another example would be to have the sub-heading as 'The Big Screen (1993-1996)'. This gets rid of some text.

The only thing that I found lacking under the Timeline/Stages section was a description of the image. I don't think a detailed descritption is necessary because the table is very well constructed. For example, state when the heart begins to develop or when it has finished developing, and place this information on the related image's page.

One of my favourite parts was the table under the Genetics section 'Mutations in Zebrafish Causing Developmental Heart Defects ' because the information is well constructed into a table format in a way that makes sense. In other words, the information is very clear. The information under 'How are mutations achieved?' was very interesting, I never before exactly thought about different ways of mutating.

I found a little problem in the first paragraph of current research section: 'Although the embryo was '''consistently''' studied '''continuously''' over the years...'. Just remove either bolded word. Some things that I think might enhance this section would be to include:
*Some links to published articles dealing with recent experimental research.
*Dates to identify when the research by The Laboratory of Molecular Genetics and Developmental Biology (China) was undertaken.
*The names of people/labortaories involved on the experiments on the gene of lissencephaly LIS1. In other words, inlude a specific example of a certain group of researchers that have utilised this gene.

And finally, the video under 'eye disorders' was interesting, relevant and informative at the same time. I liked it very much. Good work Group 3.

----

--[[User:S8600021|Mark Hill]] 01:44, 8 September 2009 (EST) A single image of a fish, well that really gets across the message of zebrafish development, interpret the information you have read and put it into a format that will interest the reader.

ZebraFish - Zebrafish are really cool...

I'm happy with zebrafish... never heard of them but they sound interesting :) what does everyone else think? ...Gaby Pinget

Great, who else is in our group then... Oh and i'm Sal by the way

Ok so i'm not sure who the other two are because i don't know your number but just for reference my number is z3218657.
I found this cool website with some pictures of Zebrafish embryo's developing... its pretty cool...

http://www.cas.vanderbilt.edu/bioimages/animals/danrer/zfish-devel.htm

and this website shows all the stages and times and such good for a timeline

http://zfin.org/zf_info/zfbook/stages/stages.html

Research Topics for this week:
- Timeline and Staging (Sal)
- History of Model Use (Gaby)
- Genetics (Bronwyn)
- Current Embryology Research (Jo)
--> Share what we've learnt with the group next session.

Hey Guys I have a lot of info... and i don't think i should post it all up here because there is a lot of stuff... Mainly all about the Staging though. I am going to put up a summary of the different stages. The website i gave you before ZFIN is the zebrafish database of model organisms. its great!

'''Zygote Period''' – Lasts for 0-0.75hours
Transition of one cell to two cells.
Fertilisation occurs activating cytoplastimic movement. The animal poles within the cell segregate the blastodisc from the yolk cytoplasm. Segregation continues into the cleavage staging.

'''Cleavage Period''' – Lasts for 0.75-2.25 hours
Transition from two cells to 128 cells
After the first cleavage the blastocysts division is approx every 15 minutes.

'''Blastula Period''' – Lasts for 2.25-5.25 hours
Transition from 128 cells to 50% epibolby
“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – Direct quote from the ZFIN website

'''Gastrula Period''' – Lasts for 5.25-10.33 hours
Transition from 50% epibolby to 1-4 somites
The gastrula period ends when epiboly is complete, and the tail bud has formed. Here each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations.

'''Segmentation period''' – Lasts from 10.33 – 24 hours
Transistion from 1-4 somites to Prim-5
Here dermis, vertebrae and skeletal muscle are formed

'''Pharyngula Period''' – Lasts from 24 – 48 hours
Transition from Prim 5 to Long-pec
The body axis begins to straighten and the fins begin to develop.

http://www.youtube.com/watch?v=0hGT667ktTw

'''Hatching Peroid''' – Last from 48 to 72hours
Transitions from Long-Pec to Protruding-mouth
In this period, primary organ systems develop and cartilage development begins.

'''Larval Period''' – Lasts from 72hours to 30days
transition from Protruding-mouth to Day 30-44
The pectoral fin continues to develop and the internal organs become more complex. Development continues.

'''Juvenille Period''' – Lasts from 30-44 days
Here adult fins and pigments as well as 12 teeth develop.

'''AdultPeriod''' – Lasts90days to 2 years
Full Breeding Adult.

http://www.youtube.com/watch?v=5ygcu9BRXI0 - Zebrafish heart beating!

http://www.youtube.com/watch?v=TbErcmhzUSY - alcohol effects on Zebrafish embryo

Websites used
http://dev.biologists.org/cgi/content/abstract/dev.022673v1

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

Hey! This is a really good site with a bunch of links relating to the different areas we have to research:
http://www.sanger.ac.uk/modelorgs/zebrafish.shtml

Hey guys.... Ive found a really good picture of the zebrafish embryo development (like the human one) but I dont know how to put it up, was thinking it might be better if it was actually on the page rather than a link to get the picture. JO

well I think that I just uploaded a picture but I have no idea where to... wow I'm so lost... Maybe that's a good thing because I have a feeling that it was copy right protected... DAMN COPY RIGHT ah ha ha

Hey all, all the websites that i have put up on this page have really good images... I have emailed the three of them and asked if it was ok if i used them to put up on here and i am awaiting a reply so
hopefully they will let us use them. Also i am having a tonne and a half of trouble uploading a InDesign Timeline image i created of the timeline and also having some issues with the net so i might
bring them to class and see if the computers there are readable other wise i am screwed and will just have to start all over again. I have a more complete Timeline and Staging format that i have
created as well. I'll put that up when i try and upload the image again. Peace. - Sal

Hey Hey So I got permission from Judy Cebra-Thomas to use all her images on her website which are each stage of development - YAY we just have to accredit them. So today apart from working on
unwinding my InDesign TImeline and putting the info up in bullets i am going to start to figure out whats happening in all the images and try an upload them onto the site so hopefully tomorrow we
will have an almost finished timeline and staging section! - Sal

Right so I've posted a link to the current sequencing status on the genetics part of our site. This updates pretty much everyday so I will be able to update ours right before submission so we have up-to-date info. This is however a minor detail so I'm writing it here to help us all remember! :) thanks

'''Hey guys!! please read this: I put up pictures which are really just to make it look pretty rather than add any info. Can you please tell me if you think they're stupid or not serious enough? I promise that I wont be offended! I understand if any of you think that we should take our assignment more seriously than that... it's just hard to find images for a history of zebrafish use!'''

I dont really get the light bulb lol. Sally were u able to get the embryo stage images as a link to the info as u wanted???? I think its a really good idea if we could do it, i think it might be a matter of asking someone who's actually good with computers to do it lol. Im still complying info for the current research section, currently in qld but i wanna have it up hopefully by this week, at least part of it anyways. Im gonna try n get some pictures but every place that ive asked think im an actual doctor lol so its been a bit hard.

Ok so not quite sure how to do the linking but i might just make it all link off the page into separate little pages... does that make sense??? Probably not but i can try explain it better on thursday. The
images take me 1.5hours each to upload because i have to make the image in indesign, Grab it and then transform the image to a PNG file as that is one that this website allows for uploading! So they
are coming up but ever so slowly! When i got permission to use the images i just told them I was a student studying Advance Science from UNSW. I was enrolled in an embryology course there where
we were doing an assignment on the Embryology of Zebrafish. Could i please use your images with referencing and a link to your webpage or document. Thats kind of what i said! i'm hoping to have it
all up and ready on thursday but might have to finish it friday night as have friends down from QLD that tend to make my place messy and have a house inspection on that ARVO!!! - Cheers SAL

Hey Sally, just so u know. I moved the websites you used to the references section so it didnt seem odd to keep it after ur info.

Hey Sally, we were thinkin about whether or not we could mix the timeline with the stages, so we thought that maybe if on the actual page we had the timeline with the pictures, then by clicking on the pictures there would be the info that u got or maybe even putting it all into a table so the pictures & info are more integrated.

Hey guys sorry i wasn't in class yesterday i had a funeral. What i was thinking of doing was actually moving all the data off the page and you click on it to go to another page which as all the info! but
the idea of clicking on the picture and going to it sounds great. One flaw is that is it ok if i use the pictures twice because i have been cutting, altering, adding text and flattening the images so that
they have a caption of what is happening. If i just cut another image to show each stage and then you click on that to go to it i think that could be cool as long as when you open the next page it still
has the information and the image with the caption of what is occurring! Hope thats ok. I will work on it all tonight and hopefully finish. Its taking a lot more time than expect sorry!Sal

hey Guys so I don't know if you guys have checked the website out yet but i have been working on it today! I continued what i was doing with the linked page and have also drawn my own diagrams
which are colour coded and easy for people to understand what is going on during the stages. I thought it looked cool. I haven't finished yet - a few more periods to go but i can't look at the screen
any more so i will do some on tuesday night! Hope thats ok. Just wondering though about the Nature article images above it kind of comes across the screen and i was wondering if we could somehow
move the image a little higher or enter down some spaces so that the timelines section is lower and the nature article doesn't come over the images pushing the table to the left! Let me know what you
all think. Sal

Hey Sal, your part of the project looks great! I really like the idea of clicking onto another page to get the info. works really really well and it's really easy to understand. As for the size of the picture obscuring your section, I think that you already thumbnailed it and it looks fine. Also, do you know how to put a youtube video onto the webpage? I was able to for a project in another class and it looked good but that was much easier because it had a link to a bunch of plug ins i could use. If anyone has any ideas please let me know! Gaby

Hey Sally, ur part looks awesome!!! Everyone done forget about your references...ive tried making the ones there so far look like it was done by one person lol. I dont know how to do the youtube clip thing but i have one too, was thinking maybe we should have a section for helpful links or do u think we should just put the links in with watever section its assiciated with. Wat do u guys think??? Jo

Hmm I was just thinking that the best thing to do was to put up the links strait after whatever they're associated with, that way they are supplimentary to what the reader is looking at rather than random extras at the end. That'll prob work best, right? Gaby

Thanks guys I've put a tonne of effort in so hope it pays off! I was going to fix up the references (cause they just have my websites without being real references) but it took me a lot longer than i thought for the images as you
can see its 5am. Nice. Yes i think linking the videos with the relevant parts is best. I have added links to videos throughout my section. Also I have made it so that when you put your mouse over the images it comes up with its own
reference/copy right info. Also when adding a link to another website its best to name the website instead of having just the web address. Its really easy if you don't know how and i can show you how to fix them up. When we get our
feedback from the other groups we can add and so forth before the project is actually marked which is really great. I think we need more info with the Genetics part but i figure bron is doing that tomorrow! Jo I really love your
sections pics and info it great and to the point!Gaby, yeah i did move it hope thats ok. I do have one question though and not meaning to hurt your feelings but i'm not that keen on the drawn image of the bird,fish,frog and mouse.
It doesn't really fit. Sorry, I mean obviously the decision is up to you, but i think it just looks a bit odd in the middle of the page because it you see it first and think , what? That was just some of the other feedback i got
from some of my friends that i asked to check out the website and see how they could maneuver around it. So that's my major spiel. Catch ya tomorrow. Sal

2009 Group Project 3

2009-10-14T04:25:33Z

Z3218657: /* References */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:24:50Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:23:55Z

Z3218657: /* Website Links */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html DB Lab: Zebrafish Staging]

[http://www.ensembl.org/Danio_rerio/Info/Index Ensembl Zebrafish: Zebrafish Danio rerio sequence]

[http://www.eusem.com/main/read-eye EU Science Education Media: Retinitis Pigmentosa]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html National Center for Biotechnology Information: Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ Sanger Institute: The Danio Rerio Sequencing Project]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Sidwell: Zebrafish Slides]

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 UCSC Genome Bioinformatics: Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:16:31Z

Z3218657: /* Website Links */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://zfin.org/zf_info/zfbook/stages/stages.html The Zebrafish Model Organism Database: Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:15:13Z

Z3218657: /* References */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Videos===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

===Website Links===

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:13:42Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) Timeline: Headwaters of the Zebrafish emergence of a new water vertebrae, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:13:09Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grunwald DJ, Eisen JS (2002) '''Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae''', Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:07:13Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald DJ, Eisen JS (2002) ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) [http://www.sciencemag.org/cgi/content/abstract/322/5904/1065 Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy] Science, 322:1065-1069.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T04:03:38Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore J (1999) [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton R, Milam J & Roe B (2001) [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman N & Yelon D (2002) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green D & Reed J (1998) [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald DJ, Eisen JS (2002) ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller P, Schmidt A, Wittbrodt J, Stelzer E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Kendrick C, Zhang L, Jülich D and Holley AS [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Department of Molecular, Cellular and Developmental Biology, Yale University, USA.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995)[http://www.ncbi.nlm.nih.gov/pubmed/8589427?dopt=Abstract Stages of Embryonic Development of the Zebrafish] Institute of Neuroscience, University of Oregon, Eugene USA. [PMID: 8589427]

Postlethwait JH (2006) [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes, 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008) [http://www.ncbi.nlm.nih.gov/pubmed/18723859 Genetic & Physical Interaction between NPHP5 and NPHP6 gene products] Renal Division, University Hospital Freiburg, Germany, 17(23):3655-62. [PMID: 18723859]

Stainier D (2001) [http://www.find-health-articles.com/rec_pub_11253067-zebrafish-genetics-vertebrate-heart-formation.htm Zebrafish Genetics and Vertebrate Heart Formation] Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Expression and Function on Embryonic Development of Lissencephaly-1 genes in Zebrafish] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China, 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) [http://www.ncbi.nlm.nih.gov/sites/entrez Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish] The Laboratory of Molecular Genetics and Development Biology, College of Life Science, Wuhan University, China, 41(8):677-88. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:44:26Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D. & Reed, J. (1998). [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). [http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstractBuch&ProduktNr=232003&ArtikelNr=95104 The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff] Vertebrate Genomes 2:183-197

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:38:59Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D. & Reed, J. (1998). [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, Nature Reviews Genetics, 3:717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate] European Molecular Biology Laboratory

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:35:46Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). [http://nar.oxfordjournals.org/cgi/content/abstract/27/8/1767 Animal mitochondrial genomes] Nucleic Acids Research, 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5):148-149.

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern University.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D. & Reed, J. (1998). [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:33:09Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5): e148

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern university.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D. & Reed, J. (1998). [https://www.sciencemag.org/cgi/content/abstract/281/5381/1309 Mitochondria and Apoptosis] Science, 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:28:05Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5): e148

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern university.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) [http://www.ncbi.nlm.nih.gov/pubmed/19757379 Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos] Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:13:34Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5): e148

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Bio-Medicine, Northwestern university.

Glickman, N., & Yelon, D. (2002). [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=12468254&ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cardiac development in zebrafish:coordination of form and function] Cell and Developmental Biology, 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) ''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''. Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:09:56Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5): e148

Broughton, R., Milam, J., & Roe, B. (2001 [http://genome.cshlp.org/cgi/content/abstract/11/11/1958 The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and Evolutionary Patterns in Vertebrate Mitochondrial DNA] Genome Research, 11:1958-1967.

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer] Northwestern university

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology, 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) ''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''. Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T03:03:01Z

Z3218657: /* Articles */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Bradbury J (2004) [http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science], PLoS Biology 2(5): e148

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Crown, Elizabeth (2005) [http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]; Northwestern university

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology, 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. (2009) ''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''. Department of Biochemistry, National University of Ireland, Ireland. [PMID: 19757379]

Heidelberg (2008) [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate],

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E. (2008) “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, ScienceExpress.

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A [http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G (2008), ''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Renal Division, University Hospital Freiburg, Germany. 1;17(23):3655-62. [PMID: 18723859]

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427]

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews, 2:39-48.

Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M (2009) ''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''.. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 41(8):677-88. [PMID-19657569]

Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. (2009) ''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. [PMID-19693699]

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-14T02:48:10Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Website Links===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004[

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

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[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004[

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:48:59Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

[http://zfin.org/zf_info/zfbook/stages/stages.html Stages of Zebrafish Development]

[http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html Zebrafish Staging]

[http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm Zebrafish Slides]

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004[

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:45:22Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

[http://www.youtube.com/watch?v=ZItgyfuxsfM (Video) Eye Disorders and Zebrafish]

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

[http://www.youtube.com/watch?v=JSMJ_47_5D4 (Video) First Digital Blueprint of Zebrafish Embryo]

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004[

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:43:11Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004[

[http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html Zebrafish Genome Resources]

[http://www.sanger.ac.uk/Projects/D_rerio/ The Danio Rerio Sequencing Project]

[http://www.ensembl.org/Danio_rerio/Info/Index Zebrafish Danio rerio sequence]

[http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5 Zebrafish (Danio rerio) Genome Browser Gateway]

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:38:27Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

[http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science] Bradbury J (2004) PLoS Biology 2(5): e148; May 11, 2004

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:35:37Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

[http://www.eusem.com/main/read-eye EUSEM EU Science Education Media]

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-12T10:33:02Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “''There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development''” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “''committed to answering a question''” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “''described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae''” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “''patterning and differentiation of the nervous system''” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “''illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo''” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “''placed the zebrafish in the context of vertebral biology''” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “''the fish is a frog... is a chicken... is a mouse''” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “''The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems''” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The Institute of Biochemistry and Cell Biology, China found that the zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified by the Department of Biochemistry, Ireland as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

The Massachusetts Institute of Technology, Boston also depicted a mutant zebrafish embryo, where the embryo was injected with a mutagenesis and subsequently resulted in the failure of the synthesis of melanin. Hence the Institute of Technology determined that the failure of the synthesis of melanin resulted in the mutant zebrafish lacking adequate black pigments in melanocytes, as depicted in the file: A Zebrafish Pigment Mutant.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. The Renal Division of the University Hospital in Freiburg, Germany determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==Glossary==

'''Antisense morpholinos''' – a molecule used to modify genetic expression

'''Blastula''' – the early embryological developmental stage consisting of a single layer of cells enclosing a cavity

'''Cleavage''' – a series of mitotic divisions by a fertilized ovum

'''Contig''' – a fragment of a DNA sequence

'''Diploid''' – a cell with double the number of chromosomes

'''Ectoderm''' – the outermost germ cell layer of an embryo

'''Endoderm''' – the innermost germ cell layer of an embryo

'''Epiboly''' – the rapid proliferation of cells

'''Gastrula''' – the early stage of germ cell formation consisting to ectoderm, mesoderm and endoderm

'''Genotype''' – the genetic makeup of an organism

'''Haploid''' – a cell with a complete set (half the number of a diploid cell) of chromosomes

'''Heterozygous''' – an organism with different alleles

'''Homozygous''' – an organism with identical alleles

'''Lissencephaly''' – an abnormality of brain formation

'''Meroblastic''' – the partial cleavage of a fertilized egg

'''Mesoderm''' – the middle germ cell layer of an embryo

'''Mutagenesis''' – the occurrence and the development of mutation

'''Nephronophthisis''' – the genetic abnormality of the kidneys

'''N-Ethyl-N-Nitrosourea''' – a mutagen

'''Oxidative Phosphorylation''' – the metabolic pathways for the production of ATP

'''Phenotype''' – the expression of specific characteristics of an organism based upon its genetic and environmental influences

'''Progeny''' – the resulting offspring

'''Retinitis Pigmentosa''' – the degeneration of the retina, resulting in visual loss

'''Situs Inversus''' – a congenital abnormality where visceral organs are inverted

'''Zygote''' – a cell the develops as a result of fertilization

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

[http://news.bio-medicine.org/biology-news-3/Tiny-zebrafish-teaches-researchers-how-to-fight-off-a-deadly-cancer-11249-1/ Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer]Crown,Elizabeth; Jun, 2005, Northwestern University Jun, 2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

http://www.eusem.com/main/read-eye EUSEM- EU Science Education Media

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020148 Small Fish, Big Science

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

Z3218657

2009-10-08T03:41:31Z

Z3218657:

'''Lab 1 Questions'''

1. What is the protein that the sperm binds to on the surface of the ova?
ZP3 protein

2. Name the 3 stages of follicle development in the ovary?
Primary
Secondary
Graafian

Though I thought i loaded this up when I was in Class... At least i thought i did sorry
and i also heard the question differently.

--[[User:Z3218657|Sally Clarke]] 13:12, 6 August 2009 (EST)
'''Lab 2 Questions'''

1.What factor do the synctiotrophoblast cells secrete to support the ongoing pregnancy? HCG (human chorionic Gonadotropin) hormone secreted to stop the menstral cycle by acting on cells in the ovary. These cells in the ovary secrete hormones such as progesterone and oestrogen to stop the menstral flow. It also maintains the Decidua and the Corpus Luteum and secretes proteletic enzymes. HCG in the urine is what a pregnacy test, tests for to determine pregancy.

2.What does the corpus luteum secrete to prevent continuation of the menstrual cycle? The Leteul cells – granulosa and theca (synthesise in the hormone) secretes Progesterone which stops menstral cycle. The corpus luteum is signalled by the HCG produced by the Synctiotrophoblasts.

3.What are the 2 main tissues to be derived from the germ cell layer continuous with the lining of the amniotic sac? The main tissues are the Nervous Tissue and Epithelial Layer

Process: Epiblast and hypoblast layers are in the Bilaminal Layers --> formation of the Trilaminal Layers with the ectoderm, mesoderm and endoderm layers. Here the Hyperblast layer is lost and the Yoke, Chorionic and Amnoitic Sac are formed.
Yoke sac – layer is lined with endoderm, contributes to the gut formation – The yoke sac is lined with the same cells that will form the gut
Corionic sac – aminon fuses with the cornion

--[[User:Z3218657|Sally Clarke]] 13:05, 13 August 2009 (EST)
'''Lab 3 Questions'''

1.What period of human development (in weeks) do the 23 Carnegie stages cover?
8weeks – 8 weeks 4 days

2.What part of the somite will contribute to the vertebral column?
The column differentiates from the paired sclerotome component (ventral half) of the somite and the midline ventral patterning structure the notochord thus contributing to the vertebral column.

3.At what Carnegie stage does the human neural tube normally completely close?
Stage 13 – 4 weeks.

--[[User:Z3218657|Sally Clarke]] 13:10, 20 August 2009 (EST)
'''Lab 4 Questions'''

1. Into what structure do most blood vessels empty before they enter the embryonic heart?
Sinus Venosus

2. What do the dorsal aortas become in the adult?
The Desending Aorta

3. What are the layers of cells found in a tertiary villi?
* Cytotrophoblast layer
* Extra-Embryonic Mesoderm
* Blood Vessels

--[[User:Z3218657|Sally Clarke]] 13:14, 27 August 2009 (EST)
'''Lab 5 Questions'''

1. What was the question I said in the respiratory lecture would be part of this week's assessment?
I'm assuming the question was to "Explain the foramen of Bochdalek within Congenital Diaphragmatic Hernia"

2. What is the answer to the above question?
When the pleuroperitoneal foramen (foramen of Bochdalek) fails to close it allows different viscera into thorax. Thus Intestine, stomach or spleen can enter the pleural cavity, compressing the lung which stops full development of the lung. This usually occurs on the left side more commonly than the right.

--[[User:Z3218657|Sally Clarke]] 13:06, 3 September 2009 (EST)
'''Lab Question 6'''

1. Which is the more common clefting, cleft lip or cleft palate?
The Cleft lip is more common because a cleft lip always forms from a cleft palate but a cleft lip can also occur individually as well, whereas a cleft palate can not.

2. What structures does pharyngeal pouch 1 form?
The Pharyngeal arch elongates to form tubotympanic recess, tympanic cavity, mastoid antrum, eustachian tube

3. Neural crest forms which cells within the skin?
Forms melanocytes within the skin

'''My apologies for my absence from class I was at a Funeral. I will complete the questions tonight. Sal'''

'''Lab 7 Questions'''

1. What is a myotube and how is it formed?
It is a multi-nucleated cell that comes from the differentiation of myoblasts and creates the mature muscle fibres.

2. What changes would I expect to see in the muscle fibre types in my leg if I...

(a) Suffered a spinal cord injury?
Muscle Atrophy would occur within the muscles

(b) Took up Marathon Running?
There would be a conversion to slow twitch fibres within the muscles

--[[User:Z3218657|Sally Clarke]] 13:57, 24 September 2009 (EST)'''Lab 8 Questions'''

Feedback on Websites

--[[User:Z3218657|Sally Clarke]] 14:29, 1 October 2009 (EST)

--[[User:Z3218657|Sally Clarke]] 14:38, 8 October 2009 (EST)'''Lab 10 Questions'''

Question 1. Identify and name 3 tissue types which contain adult (somatic) stem cells that were used/studied from the above 5 articles.
*Muscle
*Fat(adipose tissue)
*Bone Marrow

Question 2. Name 2 reprogramming strategies/methods used in generating human induced Pluripotent Stem Cells (iPSCs) from the above 5 articles.
* Virus - '''Lenti Virus''' containing 4 genesIntroduction of 4 genes into Adipose tissue,its twice as quick growth. Want to find out which type of Adipose cell is most programable. Yamanaka Factors.
* Virus Free integrations '''Epizonal Vectors'''- avoids viruses intergrating into the genome

Pluripotency is the ability of the human embryonic stem cell to differentiate or become almost any cell in the body.

Question 3. Is the following statement TRUE or FALSE?
"Unlike the nuclear genome, the mitochondrial DNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin."
*TRUE

Z3218657

2009-10-08T03:39:18Z

Z3218657:

'''Lab 1 Questions'''

1. What is the protein that the sperm binds to on the surface of the ova?
ZP3 protein

2. Name the 3 stages of follicle development in the ovary?
Primary
Secondary
Graafian

Though I thought i loaded this up when I was in Class... At least i thought i did sorry
and i also heard the question differently.

--[[User:Z3218657|Sally Clarke]] 13:12, 6 August 2009 (EST)
'''Lab 2 Questions'''

1.What factor do the synctiotrophoblast cells secrete to support the ongoing pregnancy? HCG (human chorionic Gonadotropin) hormone secreted to stop the menstral cycle by acting on cells in the ovary. These cells in the ovary secrete hormones such as progesterone and oestrogen to stop the menstral flow. It also maintains the Decidua and the Corpus Luteum and secretes proteletic enzymes. HCG in the urine is what a pregnacy test, tests for to determine pregancy.

2.What does the corpus luteum secrete to prevent continuation of the menstrual cycle? The Leteul cells – granulosa and theca (synthesise in the hormone) secretes Progesterone which stops menstral cycle. The corpus luteum is signalled by the HCG produced by the Synctiotrophoblasts.

3.What are the 2 main tissues to be derived from the germ cell layer continuous with the lining of the amniotic sac? The main tissues are the Nervous Tissue and Epithelial Layer

Process: Epiblast and hypoblast layers are in the Bilaminal Layers --> formation of the Trilaminal Layers with the ectoderm, mesoderm and endoderm layers. Here the Hyperblast layer is lost and the Yoke, Chorionic and Amnoitic Sac are formed.
Yoke sac – layer is lined with endoderm, contributes to the gut formation – The yoke sac is lined with the same cells that will form the gut
Corionic sac – aminon fuses with the cornion

--[[User:Z3218657|Sally Clarke]] 13:05, 13 August 2009 (EST)
'''Lab 3 Questions'''

1.What period of human development (in weeks) do the 23 Carnegie stages cover?
8weeks – 8 weeks 4 days

2.What part of the somite will contribute to the vertebral column?
The column differentiates from the paired sclerotome component (ventral half) of the somite and the midline ventral patterning structure the notochord thus contributing to the vertebral column.

3.At what Carnegie stage does the human neural tube normally completely close?
Stage 13 – 4 weeks.

--[[User:Z3218657|Sally Clarke]] 13:10, 20 August 2009 (EST)
'''Lab 4 Questions'''

1. Into what structure do most blood vessels empty before they enter the embryonic heart?
Sinus Venosus

2. What do the dorsal aortas become in the adult?
The Desending Aorta

3. What are the layers of cells found in a tertiary villi?
* Cytotrophoblast layer
* Extra-Embryonic Mesoderm
* Blood Vessels

--[[User:Z3218657|Sally Clarke]] 13:14, 27 August 2009 (EST)
'''Lab 5 Questions'''

1. What was the question I said in the respiratory lecture would be part of this week's assessment?
I'm assuming the question was to "Explain the foramen of Bochdalek within Congenital Diaphragmatic Hernia"

2. What is the answer to the above question?
When the pleuroperitoneal foramen (foramen of Bochdalek) fails to close it allows different viscera into thorax. Thus Intestine, stomach or spleen can enter the pleural cavity, compressing the lung which stops full development of the lung. This usually occurs on the left side more commonly than the right.

--[[User:Z3218657|Sally Clarke]] 13:06, 3 September 2009 (EST)
'''Lab Question 6'''

1. Which is the more common clefting, cleft lip or cleft palate?
The Cleft lip is more common because a cleft lip always forms from a cleft palate but a cleft lip can also occur individually as well, whereas a cleft palate can not.

2. What structures does pharyngeal pouch 1 form?
The Pharyngeal arch elongates to form tubotympanic recess, tympanic cavity, mastoid antrum, eustachian tube

3. Neural crest forms which cells within the skin?
Forms melanocytes within the skin

'''My apologies for my absence from class I was at a Funeral. I will complete the questions tonight. Sal'''

'''Lab 7 Questions'''

1. What is a myotube and how is it formed?
It is a multi-nucleated cell that comes from the differentiation of myoblasts and creates the mature muscle fibres.

2. What changes would I expect to see in the muscle fibre types in my leg if I...

(a) Suffered a spinal cord injury?
Muscle Atrophy would occur within the muscles

(b) Took up Marathon Running?
There would be a conversion to slow twitch fibres within the muscles

--[[User:Z3218657|Sally Clarke]] 13:57, 24 September 2009 (EST)'''Lab 8 Questions'''

Feedback on Websites

--[[User:Z3218657|Sally Clarke]] 14:29, 1 October 2009 (EST)

--[[User:Z3218657|Sally Clarke]] 14:38, 8 October 2009 (EST)'''Lab 10 Questions'''

Question 1. Identify and name 3 tissue types which contain adult (somatic) stem cells that were used/studied from the above 5 articles.
*Muscle
*Fat(adipose tissue)
*Bone Marrow

Question 2. Name 2 reprogramming strategies/methods used in generating human induced Pluripotent Stem Cells (iPSCs) from the above 5 articles.
* Virus - '''Lanti Virus''' containing 4 genesIntroduction of 4 genes into Adipose tissue,its twice as quick growth. Want to find out which type of Adipose cell is most programable. Yamanaka Factors.
* Virus Free integrations '''Epizonal Vectors'''- avoids viruses intergrating into the genome

Pluripotency is the ability of the human embryonic stem cell to differentiate or become almost any cell in the body.

Question 3. Is the following statement TRUE or FALSE?
"Unlike the nuclear genome, the mitochondrial DNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin."
*TRUE

Z3218657

2009-10-08T03:38:10Z

Z3218657:

'''Lab 1 Questions'''

1. What is the protein that the sperm binds to on the surface of the ova?
ZP3 protein

2. Name the 3 stages of follicle development in the ovary?
Primary
Secondary
Graafian

Though I thought i loaded this up when I was in Class... At least i thought i did sorry
and i also heard the question differently.

--[[User:Z3218657|Sally Clarke]] 13:12, 6 August 2009 (EST)
'''Lab 2 Questions'''

1.What factor do the synctiotrophoblast cells secrete to support the ongoing pregnancy? HCG (human chorionic Gonadotropin) hormone secreted to stop the menstral cycle by acting on cells in the ovary. These cells in the ovary secrete hormones such as progesterone and oestrogen to stop the menstral flow. It also maintains the Decidua and the Corpus Luteum and secretes proteletic enzymes. HCG in the urine is what a pregnacy test, tests for to determine pregancy.

2.What does the corpus luteum secrete to prevent continuation of the menstrual cycle? The Leteul cells – granulosa and theca (synthesise in the hormone) secretes Progesterone which stops menstral cycle. The corpus luteum is signalled by the HCG produced by the Synctiotrophoblasts.

3.What are the 2 main tissues to be derived from the germ cell layer continuous with the lining of the amniotic sac? The main tissues are the Nervous Tissue and Epithelial Layer

Process: Epiblast and hypoblast layers are in the Bilaminal Layers --> formation of the Trilaminal Layers with the ectoderm, mesoderm and endoderm layers. Here the Hyperblast layer is lost and the Yoke, Chorionic and Amnoitic Sac are formed.
Yoke sac – layer is lined with endoderm, contributes to the gut formation – The yoke sac is lined with the same cells that will form the gut
Corionic sac – aminon fuses with the cornion

--[[User:Z3218657|Sally Clarke]] 13:05, 13 August 2009 (EST)
'''Lab 3 Questions'''

1.What period of human development (in weeks) do the 23 Carnegie stages cover?
8weeks – 8 weeks 4 days

2.What part of the somite will contribute to the vertebral column?
The column differentiates from the paired sclerotome component (ventral half) of the somite and the midline ventral patterning structure the notochord thus contributing to the vertebral column.

3.At what Carnegie stage does the human neural tube normally completely close?
Stage 13 – 4 weeks.

--[[User:Z3218657|Sally Clarke]] 13:10, 20 August 2009 (EST)
'''Lab 4 Questions'''

1. Into what structure do most blood vessels empty before they enter the embryonic heart?
Sinus Venosus

2. What do the dorsal aortas become in the adult?
The Desending Aorta

3. What are the layers of cells found in a tertiary villi?
* Cytotrophoblast layer
* Extra-Embryonic Mesoderm
* Blood Vessels

--[[User:Z3218657|Sally Clarke]] 13:14, 27 August 2009 (EST)
'''Lab 5 Questions'''

1. What was the question I said in the respiratory lecture would be part of this week's assessment?
I'm assuming the question was to "Explain the foramen of Bochdalek within Congenital Diaphragmatic Hernia"

2. What is the answer to the above question?
When the pleuroperitoneal foramen (foramen of Bochdalek) fails to close it allows different viscera into thorax. Thus Intestine, stomach or spleen can enter the pleural cavity, compressing the lung which stops full development of the lung. This usually occurs on the left side more commonly than the right.

--[[User:Z3218657|Sally Clarke]] 13:06, 3 September 2009 (EST)
'''Lab Question 6'''

1. Which is the more common clefting, cleft lip or cleft palate?
The Cleft lip is more common because a cleft lip always forms from a cleft palate but a cleft lip can also occur individually as well, whereas a cleft palate can not.

2. What structures does pharyngeal pouch 1 form?
The Pharyngeal arch elongates to form tubotympanic recess, tympanic cavity, mastoid antrum, eustachian tube

3. Neural crest forms which cells within the skin?
Forms melanocytes within the skin

'''My apologies for my absence from class I was at a Funeral. I will complete the questions tonight. Sal'''

'''Lab 7 Questions'''

1. What is a myotube and how is it formed?
It is a multi-nucleated cell that comes from the differentiation of myoblasts and creates the mature muscle fibres.

2. What changes would I expect to see in the muscle fibre types in my leg if I...

(a) Suffered a spinal cord injury?
Muscle Atrophy would occur within the muscles

(b) Took up Marathon Running?
There would be a conversion to slow twitch fibres within the muscles

--[[User:Z3218657|Sally Clarke]] 13:57, 24 September 2009 (EST)'''Lab 8 Questions'''

Feedback on Websites

--[[User:Z3218657|Sally Clarke]] 14:29, 1 October 2009 (EST)

--[[User:Z3218657|Sally Clarke]] 14:38, 8 October 2009 (EST)'''Lab 10 Questions'''
Question 1. Identify and name 3 tissue types which contain adult (somatic) stem cells that were used/studied from the above 5 articles.
*Muscle
*Fat(adipose tissue)
*Bone Marrow

Question 2. Name 2 reprogramming strategies/methods used in generating human induced Pluripotent Stem Cells (iPSCs) from the above 5 articles.
* Virus - '''Lanti Virus''' containing 4 genesIntroduction of 4 genes into Adipose tissue,its twice as quick growth. Want to find out which type of Adipose cell is most programable. Yamanaka Factors.
* Virus Free integrations '''Epizonal Vectors'''- avoids viruses intergrating into the genome

Pluripotency is the ability of the human embryonic stem cell to differentiate or become almost any cell in the body.

Question 3. Is the following statement TRUE or FALSE?
"Unlike the nuclear genome, the mitochondrial DNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin."
*TRUE

2009 Group Project 3

2009-10-08T02:43:27Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “committed to answering a question” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “patterning and differentiation of the nervous system” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “placed the zebrafish in the context of vertebral biology” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “the fish is a frog... is a chicken... is a mouse” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. It was determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function] Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

http://www.eurekalert.org/pub_releases/2005-06/nu-tzt060205.php- Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer 20-Jun-2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

http://www.eusem.com/main/read-eye EUSEM- EU Science Education Media

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-08T02:43:00Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “committed to answering a question” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “patterning and differentiation of the nervous system” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “placed the zebrafish in the context of vertebral biology” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “the fish is a frog... is a chicken... is a mouse” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. It was determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function]Written by Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley,Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

http://www.eurekalert.org/pub_releases/2005-06/nu-tzt060205.php- Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer 20-Jun-2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

http://www.eusem.com/main/read-eye EUSEM- EU Science Education Media

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

2009 Group Project 3

2009-10-08T02:42:13Z

Z3218657: /* Websites */

[[image:Zebrafish_image.jpg‎|thumb|450px|right|Picture 1: Zebrafish]]
‎
= ZebraFish =

==Background Information==

Model organisms, particularly vertebrates, have been researched extensively in science to reach a greater understanding of human embryo development without facing the ethical issues such studies would arouse if performed on human embryos. They are used, not only in the study of development of a healthy embryo but also for the study of diseases and in particular the role genetics plays in the presence and development of such diseases.

===Why zebrafish?===

Among these models, the zebrafish is one of the most useful due to unique characteristics, which allow it to be more easily studied than others. These advantages include the high reproductive rate of the zebrafish (something which is important for the study of genetics) and in addition to this, scientists are able to maintain them in breeding condition all year round. The fertilisation of the zebrafish is an external process; this means that the gametes can be fertilized individually by scientists, allowing for the manipulation of genetic material for the purpose of study of individual genes and the roles they play. Also, the unique transparency of the zebrafish embryo allows scientists to closely observe the development of the phenotypes without disturbing the process of development, enabling observation of the development of individual embryos from beginning to end.

''"‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists."''
(Bradbury, 2004)

In the study of most vertabrate models, development must be ceased in order to dissect the embryo for study. This hinders the study of the embryo greatly, forcing scientists to effectively ‘fill in the gaps’ of information they could not observe due to the disruption of embryo development. This however, is not necessary in the study of the zebrafish due to the transparency of the embryo.

==History of Model Use==

[http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_I1.html Timeline of the development of the zebrafish as a model organism]

===In the Begining... George Streisinger (December 1927 - August 1984)===

The use of the zebrafish as a model organism began in the late 1960s when a scientist at the University of Oregon, [[File:Streisinger ------ maybe use for website.gif|thumbnail|left|George Streisinger (1927-1984), "Founding Father" of Zebrafish Developmental and Genetic Research ]][http://zfin.org/cgi-bin/webdriver?MIval=aa-labview.apg&OID=ZDB-LAB-980209-8 George Streisinger], chose to study it based not only on the many advantages stated above but also on his love for tropical fish.

Streisinger wanted to analyse mutants among the zebrafish in order to study embryological development and stated that his aim was to “study features of the organisation and embryological development of the vertebrate nervous system through the use of mutant strains” (Grunwald and Eisen, 2002). In order to do this, he decided that first the phenotypes showing rare recessive mutations must be used to reproduce heterozygous offspring. It wasn’t until 1976 that Streisinger and fellow scientist, Charline Walker managed to produce wholly or partially homozygous offspring using sperm to activate an egg but in such a way that the sperm (genetically impotent due to UV damage) did not contribute to the genetic make-up of the offspring. In order for the eggs to remain diploid without gaining an extra set of chromosomes, the second meiotic division was inhibited, giving the offspring the entire set of chromosomes needed. This allowed for much ease in mapping out genes and correlating them with phenotypes.

[[File:Kimmel.JPG|right|thumb|Professor Charles Kimmel]]
===Charles Kimmel===

Streisinger was not the only scientist working on the embryologic development of the zebrafish. Neurologist Charles Kimmel (also at Oregon) used the zebrafish to model the development of the nervous system and vertebrae and by the mid- 1970s, Kimmel uncovered the segmental structure of its brain.

===Hesitations===

Problems soon arose in the form of doubt when it came to using a zebrafish to understand human genetics. “There was little theoretical appreciation of the degree to which vastly diverged species would share the regulatory pathways that govern cell behaviour and embryonic development” (Grunwald and Eisen, 2002). At the time, there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work. This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work. According to them, as long as they were studying nerve cells, it was all relevant.

===Taking the Plunge===

The Institute of Molecular Biology at the University of Oregon seemed to nurture the gamble-like quality in Streisinger’s work and provided him with the right environment as well as facilities to develop his model. The attitude of the institute was one which encouraged personal endeavours and above all else, it was important to be “committed to answering a question” (Grunwald and Eisen, 2002).

[[File:1981 remake of Nature journal.jpg|left|thumbnail| A 1981 issue of Nature journal]]

Once a method to produce heterozygous diploids was established, non- mutant strains of zebrafish were created and then specific mutations induced to study the phenotype and genotype relations. A 1981 issue of Nature journal published the first article pertaining to the zebrafish research which outlined the methods used by Streisinger to produce the homozygous clones of the zebrafish.

Around this time, Kimmel had “described more identifiable neurons in the zebrafish than had been recognised in any other vertebrae” (Grunwald and Eisen, 2002). They also traced the growth of the axons of spinal cord motor neurons. The common ground on which Streisinger and Kimmel based their work lead to a collaborative effort to record “patterning and differentiation of the nervous system” (Grunwald and Eisen, 2002).

In 1982, Kimmel began a 10 year program to “illuminate the developmental steps that led to the origin and organisation of distinct tissue types in the zebrafish embryo” (Grunwald and Eisen, 2002). Although Streisinger died in 1984, the project went on to describe a crucial stage in embryonic development which occurred just before gastrulation. This study was groundbreaking as the step described was the one in which the entire body plan emerged for the embryo, giving future researchers a conceptual framework (or map) from which to explore and monitor the development of cells. It also “placed the zebrafish in the context of vertebral biology” (Grunwald and Eisen, 2002).

That is, when corroborated with the work of embryologists of other species, it provided common principles of development for all vertebraes, leading Kimmel to claim excitedly “the fish is a frog... is a chicken... is a mouse” (Grunwald and Eisen, 2002). This illustrates the dawning of the idea that genetic and biological organisation is the same among a great range of animals, and that by studying any one of these vertebrates, it was possible to learn more about humans.

[[File:A fish is a frog is a chicken is a mouse.jpg]]

===The Big Screen===

Through combining the use of embryological, genetic and neurological studies, the 1980s saw Kimmel and other scientists exploring various gene mutations of the zebrafish. By combing these studies with other such studies of homologous genes and their phenotypes, mutations were found to exist across a range of animals. [[File:Development.jpg|thumb|right]] For example, a zebrafish with no tail was found to have a mutation in the same gene as that of a mouse with no tail. Such findings interested Drosophila developmental geneticists and compelled them to begin a research program on the zebrafish by the late 1980s. The main aim of such a program, as led by Nusslein-Volhard (winner of a [http://nobelprize.org/nobel_prizes/medicine/laureates/1995/illpres/more-nw-disc.html Nobel Prize] for her work with the Drosophila) and Wolfgang Driever was to replicate “Drosophila screen for embryonic pattern mutants in a vertebrate” (Grunwald and Eisen, 2002). This program, later known as the ‘Big Screen’, began in 1993 and finished in 1996 and the resultant 37 papers (describing 4000 embryonic lethal mutants) were all published in [http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F4.html volume 123 of the journal Development]

The Big Screen had an enormous impact on the scientific community, pushing the zebrafish as the latest promising research model for embryology and the roles of genetics in disease and development. “The model for genetic analysis of development and physiology that had been established in Drosophila had been extended fruitfully to new vertebrate problems” (Grunwald and Eisen, 2002). The mutations described by the report were useful for the study of genetic disease processes across a range of vertebrates, including humans.

=== The Importance of Mutations ===

Around this time, a research team of undergraduate students, under the watchful eye of John Postlethwait, developed a method by which molecules across the zebrafish genome could be tagged and then used to map out genetic mutations. This development of a linkage map provided the tools needed for genetic analysis through positional cloning. Positional cloning is a process used to identify and locate a gene by first identifying its phenotype and then, using its already known approximate position on a chromosome (called the candidate region), narrowing down the position until the gene in question and its mutants are found.

With the development of this technique, the possibilities of genetic studies of zebrafish was brought to the attention of the director of the [http://www.nih.gov/science/models/zebrafish/ National Institute of Health](of the USA), Harold Varmus, who set up a project to study the genomic development of the zebrafish as advocated for by Len Zon, Marc Fishman (no, really) and Nancy Hopkins. Progress soon came about in the form of discoveries of genes and expressed sequence tags.

In the year 2000, the project to sequence the zebrafish genome was launched by the Sanger Centre. Also, [http://www.neuro.uoregon.edu/ionmain/htdocs/faculty/westerf.html Monte Westerfield]established both the ZFIN (Zebrafish Model Organism Database) project and [http://zebrafish.org/zirc/home/guide.php ZIRC] (Zebrafish International Resource Centre). Whilst ZIRC is a building at the University of Oregon in which many of the zebrafish being studied are kept, ZFIN is an internet based database which aims to be a central reference point for the studies into zebrafish embryology, linking it with other such embryological studies in other model organisms. Both ZIRC and ZFIN often work closely together.

=== Digital Zebrafish Embryo ===

In 2008 researchers at the [http://www.embl.de/aboutus/index.html EMBL] (European Molecular Biology Laboratory) generated the first ever complete developmental imprint of a vertebrae using zebrafish as the model organism. This 'digital embryo' is a model which tracks every cell (its initial position, migration and divisions) in the embryo of a zebrafish for the first 24 hours after fertilisation. Click [http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/press09oct08_l.jpg here] to view the digital zebrafish embryo montaged with microscopy data. The varying colours on the lef half of each picture (digital embryo) indicate the direction of movement of the individual cells whist the right half is the microscopy data shown at time points in development. To view the video of the reconstructed 3D model of the developing digital zebrafish embryo, click [http://www.youtube.com/watch?v=JSMJ_47_5D4 here].

==Timeline and Stages of Embryonic Development==
{| class="wikitable" style="margin: 1em auto 1em auto"
|'''Duration''' || '''Period Name''' || '''Image'''
|-
| 0 - 0.75 hrs || '''[[Zygote Period]]''' || [[File:Zygote_-_this_image.png‎|The Zygote Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 0.75 - 2.25 hrs ||'''[[Cleavage Period]]'''||[[File:Cleavage_-_user.png|The Cleavage Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 2.25 - 5.25 hrs ||'''[[Blastula Period]]''' ||[[File:Last_try_blastula1.png|The Blastula Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 5.25 - 10.33 hrs || '''[[Gastrula Period]]''' ||[[File:Gastrula.png‎|The Gastrula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 10.33 - 24 hrs ||'''[[Segmentation Period]]''' ||[[File:Segmentation .png|The Segmentation Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 24 - 48 hrs || '''[[Pharyngula Period]]''' || [[File:Pharyngula.png|The Pharyngula Period. Photo supplied by Judy Cebra-Thomas]]
|-
| 48-72 hrs ||'''[[Hatching Period]]''' ||[[File:Hatching_.png|The Hatching Period. Photo supplied by Judy Cebra-Thomas‎]]
|-
| 72 hrs - 30 Days ||'''[[Larval Period]]''' ||[[File:Larvae1.png|The Larval Period. Photo supplied by Judy Cebra-Thomas‎]]
|}

==Genetics of the ZebraFish and Embryology==

[[File:developing_zebrafish.jpg|400px|thumb|right|We can learn about embryological development and disease through genetic mutation of the zebrafish]]

The ''Danio rerio'' (zebrafish) genome has not yet been fully sequenced. It is made up of 25 pairs of chromosomes and is approximately half the length of the human genome. As of September 24, 2009, 1,543,637,863 base pairs have been sequenced of an estimated total of 2,011,175,423 bps.

Current Sequencing Status: http://www.sanger.ac.uk/Projects/D_rerio/

=====''Danio rerio'' Genome Sequencing Project=====
Zv8, the 8th integrated Whole Genome Shotgun assembly of the zebrafish was released in June, 2008 by the Wellcome Trust Sanger Institute. This project gives us a genome length of 1,481,241,295 bps and the accompanying gene sets is comprised of 24,147 genes which code proteins.

For more information on the Zv8, visit http://www.sanger.ac.uk/Projects/D_rerio/wgs.shtml.

=====''Danio rerio'' Mitochondrial DNA Sequence=====
The zebrafish mitochondrial genome has been sequenced and was found to be 16,596 base pairs in length and contain 13 protein-coding genes. The order and content of these genes are that of the common vertebrate genome.
Mitochondria are the major source of ATP in eukaryotes through oxidative phosphorylation. Mitochondria are therefore crucial for cell life but are also important in the regulation of apoptosis. The similarity between zebrafish mitochondria and that of humans makes it possible for us to use zebrafish as an accurate mitochondrial model which will allow us to study normal and pathological mitochondria.

===Genetics and Embryology===
The zebrafish provides an excellent model for the study of vertebrate development. Embryonic development of the zebrafish takes place externally and the embryo is completely transparent. Development is simple and rapid, developing to a free swimming larva 120 hours after fertilisation, with a generation time of 10-12 weeks. This means that we can learn the effects of genetic mutation in a relatively short period of time and discover how they affect development. Identification of genes can be achieved through analysis of mutant phenotype, that is, the best way to determine the role of a specific gene is to mutate that gene so that it cannot function normally and observe what affect this has on the organism. Not only can genetic studies involving gene mutations of the zebrafish allow us to identify the role of genes but they provide us with a model to study vertebrate development and various human diseases.

=====How are mutations achieved?=====
Mutagens, such as N-ethyl-N-nitrosourea (ENU), gamma rays and X-rays are used to induce a mutation in the zebrafish. Usually the zebrafish will receive multiple treatments with a mutagen during mutagenesis. A breeding scheme is then implemented to achieve a stable mutation (this usually requires at least three generations) before analysis of mutant phenotype. Genetic screening is used to help determine where the mutation occurs and if it is present. Screens commonly used in studying the zebrafish are premeiotic and postmeiotic mutagenesis and screens of haploid and diploid progeny form a given fish. Many mutagenised fish die due to environmental stress. It is important for researchers to bear this in mind when conducting these experiments and take extra care to ensure that temperature and oxygen saturation of the water is kept constant and even that noise levels are kept to a minimum so as not to jeopardise the experiment.

=====Vertebrate Heart Development=====
The zebrafish has a major advantage over previous models for the study of vertebrate heart development in that because of their small size, embryos are not wholly reliant on the cardiovascular system. They receive enough oxygen through passive diffusion to be able to survive with fairly normal function for several days without any blood circulation. This allows researchers to thoroughly analyse severe defects of the cardiovascular system. Many mutations have been identified and analysed which cause a change the development of the heart in the zebrafish. Below is a table which identifies some known mutations and resultant phenotype changes related to heart development.

{| border='1px'
|+ Mutations in Zebrafish Causing Developmental Heart Defects
!Mutation !!Gene !!Phenotype !!When is Mutation First Observed
|-
| bonnie and clyde
| bon
| Cardia bifida
| Blastula
|-
| casanova
| Unknown
| Cardia bifida
| Blastula
|-
| two-of-hearts
| Unknown
| Cardia bifida
| Mid-somitogenesis
|-
| heart-and-soul
| Unknown
| Defects in heart tube formation
| Mid-somitogenesis
|-
| open-eyed pinhead
| oep
| Absence or reduction of nkx2.5 expression, cardia bifida, reduction of cardiac ventricular tissue
| Blastula
|-
| tell-tale-heart
| isl
| Contractility defect
| Mid-somitogenesis
|-
| silent heart
| Unknown
| Prevents contraction
|
|-
|}
Adapted from: Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48. and Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental Biology , 13:507-513.

==Current Embryology Research==

The most commonly used organism for studies of development and function is the zebrafish embryo. The zebrafish embryo has been observed for many years, from 1980s to 2009. Although the embryo was consistently studied continuously over the years, it was most commonly utilized in 2004-2006 from the use of the zebrafish in drug testing to fighting cancer to being the first blueprint of a vertebrae embryo. [http://www.youtube.com/watch?v=JSMJ_47_5D4] Recent developments have allowed for further understand about genetics; expression and function, eye disorders, organ and vertebrae development as well as reproductive studies. Recent studies utilize the zebrafish embryo due to its rapid proliferation rate and the transparency of the embryo.

===Genetics===
One of the most significant characteristics of the embryo is the notochord, a midline structure required for skeletal development of the vertebrae and as the main central structure through which the embryo develops. The Laboratory of Molecular Genetics and Developmental Biology, China isolated a contig of expressed sequence tags from the zebrafish ovary. This cDNA sequence was cloned and expressed within the oocyte, which further progressed through the cleavage stage to the blastula stage. At gastrulation, the specific gene sequence was evident in the dorsal region, restricted to the notochord and pectoral fin, during embryonic development at 48 and 72 hours. Hence the zebrafish notochord related gene (znrg) was established due to its abundant availability in the notochord, which was determined to play a vital role in notochord development within the zebrafish.

===Expression & Function===
The zebrafish has been utilized to express the gene of lissencephaly LIS1, which is a severe disease associated to brain malformation. The zebrafish exhibits two LIS1 genes; LIS1a and LIS1b. Proliferation and migration deficiencies in neural and brain development arise when the LIS1 gene is mutated or depleted. It was determined that the LIS1 genes were present within the protein and genetic structures and were expressed during embryonic development, within mature tissue samples. The LIS1 gene was predominately evident within the brain tissue and when the LIS1 gene was destroyed, it resulted in developmental deficiencies such as brain malformation, circulation and structural abnormalities. This study determined that the LIS1 gene plays a vital role in embryonic development, especially in relation to brain formation.

===Embryonic Growth & Development===
An insulin-like growth factor (IGF) has been identified as playing an essential role in normal embryonic growth and development throughout vertebrate evolution. However the specific role that the IGF family plays in embryonic development has previously been unclear. The National University of Ireland focused on the role of IGF2 in the development of the zebrafish embryo. Two genes, IGF2a & IGF2b, were identified in the zebrafish genome and modified by the introduction of antisense morpholinos. It was identified that neural and cardiovascular abnormalities arose from defects in IGF2 embryos, where embryos were ventralized and subsequently resulted in reduced growth, compact eyes and disruptions to the brain structure and cardiovascular system, especially in relation to the cardiac outflow tract, cardiac looping and cardiac valve development. There were also substantial affects to the development of anterior neural structures and the regulation of genes for dorsal-ventral patterning. Therefore it was determined through the utilization of the zebrafish embryo, that IGF2a & IGF2b played a vital role in embryonic development, especially in relation to neural and cardiovascular development.

===Cancer Research===
[[File:Mutant_embryo.png|200px|thumb|right|A Zebrafish Pigment Mutant.]]
In 2005, under the guidance of Mary J.C. Hendrix from the Northwestern University Feinburg School of Medicine, Illinois, a laboratory implanted the embryo of a zebrafish with human melanoma cells; these cells divided but did not express themselves as tumors within the embryo. They were, however maintained as plastic phenotypes, expressing specific cell types, including endothelial, neural and stem cells. Hendrix stated that cancer cells are unspecific, plastic phenotypes that are similar to embryonic stem cells. Due to the ability of the zebrafish embryo to maintain the plastic phenotype and to suppress the tumor from forming in the microenvironment, Hendrix hypothesized that there may be a causal relationship between the formation of cancer cells and the role of the environment. Hendrix therefore concluded that further study into the factors that suppressed the formation of tumor cells in the zebrafish, could potentially reverse the phenotype of tumor cells.

===Eye Disorders===
Nephronophthisis (NPHP) is a cystic kidney disease, caused by mutations of nine genes and is also associated to cerebellar defects, situs inversus and retinitis pigmentosa, where the mutation of NPHP5 and NPHP6 is associated to progressive blindness. It was determined that gene products, such as nephrocystin5 and nephrocystin6, contribute in maintaining photoreceptor homeostasis. Zebrafish embryos were utilized to analyze the genetic interaction of the gene products, once NPHP5 and NPHP6 was depleted.
The following link provides further information about the usefulness of the zebrafish as a model for eye disorder research. http://www.youtube.com/watch?v=ZItgyfuxsfM

==References==

===Websites===

[http://dev.biologists.org/cgi/content/abstract/135/12/2065 Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function, Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley*
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
]

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html

http://classic.sidwell.edu/us/science/21bio/new/staging_files/v3_document.htm

http://www.eurekalert.org/pub_releases/2005-06/nu-tzt060205.php- Tiny Zebrafish teaches Researchers how to Fight off a Deadly Cancer 20-Jun-2005

http://www.youtube.com/watch?v=ZItgyfuxsfM Eye Disorders and Zebrafish

http://www.eusem.com/main/read-eye EUSEM- EU Science Education Media

http://www.youtube.com/watch?v=JSMJ_47_5D4 First Digital Blueprint of Zebrafish Embryo.

http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html

http://www.sanger.ac.uk/Projects/D_rerio/

http://www.ensembl.org/Danio_rerio/Info/Index

http://genome.ucsc.edu/cgi-bin/hgGateway?db=danRer5

===Articles===

''Stages of embryonic development of the zebrafish'' ; [PMID: 8589427] [PubMed - indexed for MEDLINE]

''Znrg, a novel gene expressed mainly in the developing Notochord of Zebrafish''. Zhou Y, Xu Y, Liu Y, Zhang Z and Deng F. The Laboratory of Molecular Genetics and Development Biology; College of Life Science, Wuhan University, China. 2009 Aug 20. [PMID-19693699]

''Expression and Function on embryonic development of Lissencephaly-1 genes in Zebrafish''. Sun C, Xu M, Xing Z, Wu Z, Li Y, Li T and Zhao M. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Shanghai, China. 2009 Aug; 41(8):677-88. [PMID-19657569]

''Genetic & Physical Interaction between NPHP5 and NPHP6 gene products''. Schafer T, Putz M, Lienkamp S, Ganner A, Bergbreiter A, Ramachandran H, Gieloff V, Gerner M, Mattonet C, Czarnecki PG, Sayer JA, Otto EA, Hildebrandt F, Kramer-Zucker A and Walz G. Renal Division, University Hospital Freiburg, Germany. 2008 Dec 1;17(23):3655-62. [PMID: 18723859]

Grundwald, D. J., Eisen, J. S., 2002, ‘Timeline: Headwaters of the Zebrafish- emergence of a new water vertebrae’, ''Nature Reviews Genetics'', no. 3, pp 717-724.

Heidelberg,9 October 2008, ''Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate'', viewed at http://www.embl.de/aboutus/communication_outreach/media_relations/2008/081009_heidelberg/index.html on 20th September, 2009

''Insulin-like Growth factor-2 Regulates early Neural and Cardiovascular system development in Zebrafish embryos.''.Hartnett L, Glynn C, Nolan CM, Grealy M and Byrnes L. Department of Biochemistry, National University of Ireland, Ireland. 2009 Sept 4. [PMID: 19757379]

Keller, P., Schmidt, A., Wittbrodt J., Stelzer, E.; Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy, ScienceExpress, 9 October 2008

Boore, J. (1999). Animal mitochondrial genomes. Nucleic Acids Research , 27:1767-1780.

Broughton, R., Milam, J., & Roe, B. (2001). The Complete Sequence of the Zebrafish (Danio rerio) Mitochondrial Genome and

Evolutionary Patterns in Vertebrate Mitochondrial DNA. Genome Research , 11:1958-1967.

Glickman, N., & Yelon, D. (2002). Cardirac development in zebrafish:coordination of form and function. Cell and Developmental
Biology , 13:507-513.

Green, D., & Reed, J. (1998). Mitochondria and apoptosis. Science , 281:1309-1312.

Postlethwait, J. H. (2006). The Zebrafish Genome: A review and msx Gene Case Study. In J. N. Volff, Vertebrate Genomes (pp. 183-197). Oregon, USA: Karger.

Stainier, D. (2001). Zebrafish Genetics and Vertebrate Heart Formation. Nature Reviews , 2:39-48.

{{Template:Projects09}}
[[Category:Zebrafish]]

Talk:2009 Group Project 3

2009-10-05T04:48:23Z

Z3218657: /* Project Updates */

== Project Updates ==
--[[User:Z3283499|Antonio Lee]] 10:55, 2 October 2009 (EST) Hi everyone, I will be working with you during the Lab10 Tutorial and here is the news link and PDF of the manuscript for your group exercise. I encourage you to read the paper before the tutorial. Also, please indicate next to the questions below (using either your initials or student number) which one of the four questions you wish to address.
:'''Group 3 :''' [http://www.myelomabeacon.com/news/2009/09/21/study-shows-mozobil-induces-mobilization-of-stem-cells-but-not-tumor-cells/ '''Study Shows Mozobil Induces Mobilization Of Stem Cells But Not Myeloma Tumor Cells'''] in The Myeloma Beacon Published online 21 September 2009 10:28 pm [[Media:ANAT2341_Lab10_2009_Group 3 Reading.pdf|Manuscript (PDF): Safety and efficacy assessment of plerixafor in patients with multiple myeloma proven or predicted to be poor mobilizers, including assessment of tumor cell mobilization]]

:Question 1. What is the background to the existing problem / disease condition? 3220040
:Question 2. What approach / method did the research team take to tackle / improve the problem? 3218657
:Question 3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance?
:Question 4. What are the next steps in moving forward? What are the next or new hurdles to overcome?

Glossary (jo)

Referencing (sally)

Spell check/grammar (bronwyn)

Pictures - delete some, add relevant ones

research paper linkages - more maybe (everyone)

Format of overall page

Condensing text??? (possibly no condensing required)

==Constructive Criticism of Peers==

--[[User:Z3258567|Sando Rashed]] 09:06, 1 October 2009 (EST)hey good work with the page, lots of good pieces of information, i really like the timeline and stages of the embryonic development but you might want to add just a few words to each stage just to make the page suitable for all people to read (someone with no background will look at it and understand it), with your background info i like the idea but there isnt really a sentence structure, you might want to put it in dot form as that what is sounds like when reading it. you might want to add a glossary to the bottom of the page and add words that are unknown to people to make the reading more comprehensive. you might want to add some more photos to the current research just to make that part of the assignment a bit more appealing as there is a lot of information to grasp there. with your referencing, it is incorrent format you might want to go on the internet and look up harvard referencing (http://www.unisa.edu.au/ltu/students/study/referencing/harvard.pdf)but overall good job :D

--[[User:Z3218146|Julianna Lam]] 01:22, 1 October 2009 (EST)

- the history part has way too much information. it needs to be more concise.

-the images used for staging and timeline is awesome. set out very cleary and very easy to read and understand

- the mutation table is a very good idea although you can probably fix it up a bit so the information doesnt look as crammed up.

- great current research!

--[[User:Z3252231|Angama Yaquobi]] 20:48, 30 September 2009 (EST)

Congrats everyone, your team has done a great job. The project looks amazing.
*The info in the introduction is very informative especially under the heading of "Why Zebrafish".
*The images used in the timeline and stages of embryonic development is awesome and clicking on the period name gives alot of great information. It also makes the webpage look very nice and tidy- its structed very well.
*The section for genetics was very well informative especially comparing the zebrafish genome with human genome.
*I would recommend to add more images related to each section to make the webpage look more visually eye-catchy.
Overall it was a great effort by all the team members.well done.

--[[User:Z3186093|Jenny Guy]] 18:30, 30 September 2009 (EST)

Improvements:
* Some of your sentences sound as though you've cut and pasted them from websites. For example, do you guys really used "combing" in your vocabulary.
* The history goes more into finding out about the scientists instead of what they fully achieved. Try focus on that instead of the scientists preferences etc.
* Throughout the entire page there isn't much referencing at all! You'll have many paragraphs which dont refer to any article/text. You either need to include referencing in your text or at least place an number in reference to where you found out that information (corresponding with your bibliography).
* You need to reference your "websites" in the bibliography. By typing the url you are not referencing. Most of the articles are referenced so make sure you use the same system with the websites.
* You used youtube??? oh dear. I can understand you using it to gain an understanding but that cannot be used as a reference. seriously, anyone can upload it. So instead find out WHO uploaded it and find out if they published it on a RELIABLE website. Copyright?
* One of the outcomes Mark has asked for is a list of "links to related resources/research laboratories?" Where is this?

--[[User:Z3255007|Sadaf Masood]] 15:29, 30 September 2009 (EST)Hey Group 3! Congrats on your project guys, well done!
Ill just list down few things that i liked and what could be done better:

1. Needs glossary guys, so some of the scientific jargon can be understood easily

2. Your timeline is excellent, it has great pictures, maybe a little description along with it might make it more clear.

3. The project only has four sections, i m sure we need to do a bit about fertilization and detalied embryonic steps as well

4. References to specific articles are provided which is very informative if anyone wants to do an in-depth analysis.

5. Formatting is impressive and page layout is great!

overall, this is an awesome project which is simple and straight-forward. Great work guys! And Best of Luck!!!

--[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

Group 3 well done impression effort , resulting in a great formatted page, which summarises the requirements of the outcomes needed, it was straight to the point and briefly covered the main topic in regards to the zebrafish.

- The assignment has come together extremely well only a few minor edit here and there needed.

- The illustrations regarding (Timeline and Stages of Embryonic Development) are great, it extremely enhances the wiki page assignment allowing the readers to visualize the information which stand out in regard to the information it come along great.
The links to specfic research laboratories have been made throughout the information giving the reader additional information to learn more on the topic which is a great reference.

Suggestions to improve the assignment:

1. illustrations such as :"Development.jpg" are should be replace which more appropriate images which are more relevant with the topic and the illustrations should be more regarded in the information for the reader to relate and understand visually.

2. The Genetics information should include specfic genetic and chromosomal imagery revealing the structure of chromosome in comparison to humans and should identify why they are different.

3. Current research information should reveal a brief explanation on how zebrafish research has impacted human embryology and how it used and why it used to improve or help develop research programs.

4. Referencing is a problem, specfic referencing is needed as the university has guidelines. Visit this webpage I think it will help: www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

5. Glossary will also help the readers to understand specfic terms and make the assignment flow better.

Impressive wiki page, minor editing should be undertaken but overall the outcome were covered. --[[User:Z3295026|Joe Nassif]] 17:36, 29 September 2009 (EST)

--[[User:Z3217015|Mitchell Mathieson]] 09:42, 25 September 2009 (EST)Overall very very nice. Was nice and succinct, and easy to read. The information was relevant, and the current research interesting. Maybe the stages on different pages was a bit difficult to read; I would have liked to have seen at least a bit of information on the main page, which expands to more on your secondary page. The formatting of the references I think needs to be looked at maybe.

--[[User:Z3224449|Elide Newton]] 14:38, 26 September 2009 (EST) HELLO GROUP 3: Congratulations on a great assignment. Each section is equally proportioned, and well represented visually. My one point of constructive criticism would be to the section on current research. Your information is great, as it shows how the zebrafish is being used as a model for different areas of research. The one thing which would improve this would be to include the dates of these research papers, just so the reader can know how recent the work is. maybe introduce the research as Person et al (2009) has used the zebrafish to.... Also are there any pictures on this research? Just because research language is so technical, some pictures would break it up. Hope this helps you group 3.

--[[User:Z3126328|Jin Lee]] 16:06, 26 September 2009 (EST) congratulations to Group3! this is a really great assignment. overall, all the sections are well represented visually.
I liked the history part mostly! easy to read! Maybe the current research section can be improved by adding some images and relevant links. I found zebrafish is very interesting!thank you

--[[User:Z3187802|Vishnnu Shanmugam]] 19:32, 26 September 2009 (EST)Well done Zebra fish group. You've put together a well balanced assignment. The images under "Timeline and Stages of Embryonic Development" are impressive, really make the assignment stand out and the text flows nicely. I also noticed the links to researchers and research laboratories have been made throughout the text and because they are under specific headings, the reader will know what the information in the link will focus on. Additions to improve the assignment:

- there is some irrelevant images in the the text; the images "A 1981 issue of Nature journal" & "Development.jpg" are Unnecessary and could be replaced with others that support the text or summarize complex processes. (eg. motor neuron development in zebrafish or a time line showing the evolution in the use of zebrafish). The second image "Development.jpg" is better replaced with the image "A Zebrafish Pigment Mutant" as it is relevant to the text. The current research does not mension or explain the Zebrafish Pigment Mutant and thus is Unnecessary there.

- The Genetics section could do with an image of zebrafish chromosomes. This can be a simple hand drawn diagram and can be compared to the human chromosomal makeup.

- The current research section could use a description of how zebrafish research has impacted human embryology.

-The assignment needs to be properly referenced. see www.lc.unsw.edu.au/onlib/ref_apa.html for help with APA referencing.

- A Glossary would also complement the text.

Overall very impressive, only needs minor editing.

-[[User:Z3252340|Emily Wong]] 11:04, 27 September 2009 (EST) To begin with, you have done a great job group 3. It is a well researched, structured and organized page. The student contribution to the work is fairly even. The condensation of the written content is done well, with the combining of the timeline and stages in one section and extensive use of visual representation. However, information is not provided when clicking on the pictures as to what is in them. A description or explanation of what is occurring in each stage or at each time point would improve this project. Some of the images used have not been referenced properly in the image pages. Referencing is good and an extensive reference list has been provided. The content provided is very detailed by still brief and relevant to each section. This project could also be improved by adding some examples of current research and also referencing information throughout the written content. Perhaps an inclusion of a Pubmed search link in the current research section would be a good idea so that viewers can see what other research has been done using the zebrafish.

--[[User:Z3126345|Gang Liu]] 16:17, 27 September 2009 (EST)This is a very informative, well constructed and concise wikipage. It has demonstrated consistency throughout the page. There are a number of major subheadings have been included history, timeline, development, genetics and current embryology research as well as hand drawing. In addition, it has demonstrated extended research of literatures. For example, by dividing the history section in a number of subheadings such as "in the beginning", "Charles Kimmel", "Hesitations", etc. This has indicates the in-depth understanding of zebrafish embryo model use. It allows the reader to appreciate story behind the experiment, as well as scientists' thinking process and thoughts. "...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with. This put much strain on the funding given to Streisinger and the credibility of his work...".

The graphic delivery of timeline and stages are very self-explanatory and powerful. A picture explains a thousand words. It makes the section easy to follow, and interesting to read also.

However, this project can be improved by considering the following points.

*I found genetic and current embryology section were very lengthy and wordy, and lack of pictures. In addition, referencing in these two sections were inconsistent with previous sections;

*Lack of glossary list. Such as "oxidative phosphrylation", "N-ethyl-N-nitrosourea (ENU)", "Diploid" and "Haploid" need to provide meanings;

*Inconsistent referencing. Name of author need to keep in alphabetical order. Also articles and web based materials are mixed. The first five references were inconsistent with the rest in style;

Last few words. I enjoyed reading it, learned something out of it. Big thumb up for me.

--[[User:Z3217686|Thomas Dangerfield]] 13:54, 28 September 2009 (EST)Well done guys! Nice, visually stimulating assignment! Just a few points about possibly having a little more written info on the timeline, just to help explain what is in each image. Also some pictures of the chromosomes of the zebrafish would be a handy visual tool. This may just be a little picky, but i notice that you have a few heading issues:

- Beginning not Begining. (In the begining..)

- Genetics of the Zebrafish and Embryology, and the you have a 'Genetics and Embryology' a couple of headings down which seems a little unnecessary.

Just as a hint on being thorough, maybe a glossary could have been included, as well as formatting for your references. Still, a well presented assignment has been done here!

--[[User:Z3185685|Sumaiya Rahman]] 16:06, 28 September 2009 (EST) Hey guys. Well done on the assignment overall. It was interesting to read about the zebrafish. The page was well introduced. It is good that you broke up the information using subheadings. This makes the page a lot easier to read as it breaks up the big slabs of text. I think it has been mentioned, but there are a couple of spelling mistakes. Also the sentence “This however did not affect Neurologists such as Kimmel as they were not much worried about the relevance of their work” needs to be fixed up. This is only a minor error. The history section could look a little bit less messy if the pictures were placed on the same side. “The fish is a frog... is a chicken... is a mouse” picture is very cute! But maybe a little bit too big because we need to scroll across to see it (or is that just me?). You have some great references and links and a great timetable and stages section with good visuals. The genetics and current research sections have a lot of text, maybe finding a way of making these sections more visually appealing would help the readers. Also including a glossary would help. Overall nice work!

--[[User:Z3215682|Carly Mooney]] 11:43, 29 September 2009 (EST)
Impressive, it looks as though much time and effort went into making the page easy to read yet informative.A few suggestions:
*some of the images I thought weren't entirely relevant, e.g.A 1981 issue of Nature journal" & "Development.jpg". maybe even crop it so that it is just the covers photograph in the image, rather than the entire journal.
*Maybe decrease the size of the fish is a frog is a chicken is a mouse jpg
*Maybe move the genetics picture further down as it is very similar and close to the timeline and stages section
*I think a glossary may help
*The references to be referenced properly, and the articles should have the pubmed number with their reference
*a few grammatical errors that need to be fixed up
These are all minor thoughts, well done.

--[[User:Z3254857|Begum Sonmez]] 14:53, 30 September 2009 (EST)
Hey Group 3. Let me start off by saying that I found your project on the Zebrafish unique. Under the history section:
*There were some sentences that were unnessesary such as 'Streisinger was not the only scientist working on the embryologic development of the zebrafish.'

*It was different and iteresting to learn about the different views of Streisinger and Kimmel. I enjoyed that fact that the structure wasn't entirely date than author than finding, but the inclussion of 'disputes and uncertainties' ('...there was no gene cloning and little understanding about genes, making the whole zebrafish project almost a gamble to follow through with.') as well.

*The information flows from Streisinger to Kimmel to hesitations.

*There is too much text bunched up under the sub-headings 'Taking the plunge', 'The Big Screen', and 'The Importance of Mutations'. Do not delete any text because I found it very interesting, but I suggest the use of Sub-sub headings, or some dot points. Another example would be to have the sub-heading as 'The Big Screen (1993-1996)'. This gets rid of some text.

The only thing that I found lacking under the Timeline/Stages section was a description of the image. I don't think a detailed descritption is necessary because the table is very well constructed. For example, state when the heart begins to develop or when it has finished developing, and place this information on the related image's page.

One of my favourite parts was the table under the Genetics section 'Mutations in Zebrafish Causing Developmental Heart Defects ' because the information is well constructed into a table format in a way that makes sense. In other words, the information is very clear. The information under 'How are mutations achieved?' was very interesting, I never before exactly thought about different ways of mutating.

I found a little problem in the first paragraph of current research section: 'Although the embryo was '''consistently''' studied '''continuously''' over the years...'. Just remove either bolded word. Some things that I think might enhance this section would be to include:
*Some links to published articles dealing with recent experimental research.
*Dates to identify when the research by The Laboratory of Molecular Genetics and Developmental Biology (China) was undertaken.
*The names of people/labortaories involved on the experiments on the gene of lissencephaly LIS1. In other words, inlude a specific example of a certain group of researchers that have utilised this gene.

And finally, the video under 'eye disorders' was interesting, relevant and informative at the same time. I liked it very much. Good work Group 3.

----

--[[User:S8600021|Mark Hill]] 01:44, 8 September 2009 (EST) A single image of a fish, well that really gets across the message of zebrafish development, interpret the information you have read and put it into a format that will interest the reader.

ZebraFish - Zebrafish are really cool...

I'm happy with zebrafish... never heard of them but they sound interesting :) what does everyone else think? ...Gaby Pinget

Great, who else is in our group then... Oh and i'm Sal by the way

Ok so i'm not sure who the other two are because i don't know your number but just for reference my number is z3218657.
I found this cool website with some pictures of Zebrafish embryo's developing... its pretty cool...

http://www.cas.vanderbilt.edu/bioimages/animals/danrer/zfish-devel.htm

and this website shows all the stages and times and such good for a timeline

http://zfin.org/zf_info/zfbook/stages/stages.html

Research Topics for this week:
- Timeline and Staging (Sal)
- History of Model Use (Gaby)
- Genetics (Bronwyn)
- Current Embryology Research (Jo)
--> Share what we've learnt with the group next session.

Hey Guys I have a lot of info... and i don't think i should post it all up here because there is a lot of stuff... Mainly all about the Staging though. I am going to put up a summary of the different stages. The website i gave you before ZFIN is the zebrafish database of model organisms. its great!

'''Zygote Period''' – Lasts for 0-0.75hours
Transition of one cell to two cells.
Fertilisation occurs activating cytoplastimic movement. The animal poles within the cell segregate the blastodisc from the yolk cytoplasm. Segregation continues into the cleavage staging.

'''Cleavage Period''' – Lasts for 0.75-2.25 hours
Transition from two cells to 128 cells
After the first cleavage the blastocysts division is approx every 15 minutes.

'''Blastula Period''' – Lasts for 2.25-5.25 hours
Transition from 128 cells to 50% epibolby
“Epiboly, beginning in the late blastula (Solnica-Krezel and Driever, 1994), is the thinning and spreading of both the YSL and the blastodisc over the yolk cell, as you might model by pulling a knitted ski cap over your head” – Direct quote from the ZFIN website

'''Gastrula Period''' – Lasts for 5.25-10.33 hours
Transition from 50% epibolby to 1-4 somites
The gastrula period ends when epiboly is complete, and the tail bud has formed. Here each germ layer (endoderm, mesoderm, ectoderm) is put in the right place so that bodily organs and tissues can form in the correct locations.

'''Segmentation period''' – Lasts from 10.33 – 24 hours
Transistion from 1-4 somites to Prim-5
Here dermis, vertebrae and skeletal muscle are formed

'''Pharyngula Period''' – Lasts from 24 – 48 hours
Transition from Prim 5 to Long-pec
The body axis begins to straighten and the fins begin to develop.

http://www.youtube.com/watch?v=0hGT667ktTw

'''Hatching Peroid''' – Last from 48 to 72hours
Transitions from Long-Pec to Protruding-mouth
In this period, primary organ systems develop and cartilage development begins.

'''Larval Period''' – Lasts from 72hours to 30days
transition from Protruding-mouth to Day 30-44
The pectoral fin continues to develop and the internal organs become more complex. Development continues.

'''Juvenille Period''' – Lasts from 30-44 days
Here adult fins and pigments as well as 12 teeth develop.

'''AdultPeriod''' – Lasts90days to 2 years
Full Breeding Adult.

http://www.youtube.com/watch?v=5ygcu9BRXI0 - Zebrafish heart beating!

http://www.youtube.com/watch?v=TbErcmhzUSY - alcohol effects on Zebrafish embryo

Websites used
http://dev.biologists.org/cgi/content/abstract/dev.022673v1

http://www.zfic.org/classroom%20experiments/stagingindex.html

http://zfin.org/zf_info/zfbook/stages/stages.html

Hey! This is a really good site with a bunch of links relating to the different areas we have to research:
http://www.sanger.ac.uk/modelorgs/zebrafish.shtml

Hey guys.... Ive found a really good picture of the zebrafish embryo development (like the human one) but I dont know how to put it up, was thinking it might be better if it was actually on the page rather than a link to get the picture. JO

well I think that I just uploaded a picture but I have no idea where to... wow I'm so lost... Maybe that's a good thing because I have a feeling that it was copy right protected... DAMN COPY RIGHT ah ha ha

Hey all, all the websites that i have put up on this page have really good images... I have emailed the three of them and asked if it was ok if i used them to put up on here and i am awaiting a reply so
hopefully they will let us use them. Also i am having a tonne and a half of trouble uploading a InDesign Timeline image i created of the timeline and also having some issues with the net so i might
bring them to class and see if the computers there are readable other wise i am screwed and will just have to start all over again. I have a more complete Timeline and Staging format that i have
created as well. I'll put that up when i try and upload the image again. Peace. - Sal

Hey Hey So I got permission from Judy Cebra-Thomas to use all her images on her website which are each stage of development - YAY we just have to accredit them. So today apart from working on
unwinding my InDesign TImeline and putting the info up in bullets i am going to start to figure out whats happening in all the images and try an upload them onto the site so hopefully tomorrow we
will have an almost finished timeline and staging section! - Sal

Right so I've posted a link to the current sequencing status on the genetics part of our site. This updates pretty much everyday so I will be able to update ours right before submission so we have up-to-date info. This is however a minor detail so I'm writing it here to help us all remember! :) thanks

'''Hey guys!! please read this: I put up pictures which are really just to make it look pretty rather than add any info. Can you please tell me if you think they're stupid or not serious enough? I promise that I wont be offended! I understand if any of you think that we should take our assignment more seriously than that... it's just hard to find images for a history of zebrafish use!'''

I dont really get the light bulb lol. Sally were u able to get the embryo stage images as a link to the info as u wanted???? I think its a really good idea if we could do it, i think it might be a matter of asking someone who's actually good with computers to do it lol. Im still complying info for the current research section, currently in qld but i wanna have it up hopefully by this week, at least part of it anyways. Im gonna try n get some pictures but every place that ive asked think im an actual doctor lol so its been a bit hard.

Ok so not quite sure how to do the linking but i might just make it all link off the page into separate little pages... does that make sense??? Probably not but i can try explain it better on thursday. The
images take me 1.5hours each to upload because i have to make the image in indesign, Grab it and then transform the image to a PNG file as that is one that this website allows for uploading! So they
are coming up but ever so slowly! When i got permission to use the images i just told them I was a student studying Advance Science from UNSW. I was enrolled in an embryology course there where
we were doing an assignment on the Embryology of Zebrafish. Could i please use your images with referencing and a link to your webpage or document. Thats kind of what i said! i'm hoping to have it
all up and ready on thursday but might have to finish it friday night as have friends down from QLD that tend to make my place messy and have a house inspection on that ARVO!!! - Cheers SAL

Hey Sally, just so u know. I moved the websites you used to the references section so it didnt seem odd to keep it after ur info.

Hey Sally, we were thinkin about whether or not we could mix the timeline with the stages, so we thought that maybe if on the actual page we had the timeline with the pictures, then by clicking on the pictures there would be the info that u got or maybe even putting it all into a table so the pictures & info are more integrated.

Hey guys sorry i wasn't in class yesterday i had a funeral. What i was thinking of doing was actually moving all the data off the page and you click on it to go to another page which as all the info! but
the idea of clicking on the picture and going to it sounds great. One flaw is that is it ok if i use the pictures twice because i have been cutting, altering, adding text and flattening the images so that
they have a caption of what is happening. If i just cut another image to show each stage and then you click on that to go to it i think that could be cool as long as when you open the next page it still
has the information and the image with the caption of what is occurring! Hope thats ok. I will work on it all tonight and hopefully finish. Its taking a lot more time than expect sorry!Sal

hey Guys so I don't know if you guys have checked the website out yet but i have been working on it today! I continued what i was doing with the linked page and have also drawn my own diagrams
which are colour coded and easy for people to understand what is going on during the stages. I thought it looked cool. I haven't finished yet - a few more periods to go but i can't look at the screen
any more so i will do some on tuesday night! Hope thats ok. Just wondering though about the Nature article images above it kind of comes across the screen and i was wondering if we could somehow
move the image a little higher or enter down some spaces so that the timelines section is lower and the nature article doesn't come over the images pushing the table to the left! Let me know what you
all think. Sal

Hey Sal, your part of the project looks great! I really like the idea of clicking onto another page to get the info. works really really well and it's really easy to understand. As for the size of the picture obscuring your section, I think that you already thumbnailed it and it looks fine. Also, do you know how to put a youtube video onto the webpage? I was able to for a project in another class and it looked good but that was much easier because it had a link to a bunch of plug ins i could use. If anyone has any ideas please let me know! Gaby

Hey Sally, ur part looks awesome!!! Everyone done forget about your references...ive tried making the ones there so far look like it was done by one person lol. I dont know how to do the youtube clip thing but i have one too, was thinking maybe we should have a section for helpful links or do u think we should just put the links in with watever section its assiciated with. Wat do u guys think??? Jo

Hmm I was just thinking that the best thing to do was to put up the links strait after whatever they're associated with, that way they are supplimentary to what the reader is looking at rather than random extras at the end. That'll prob work best, right? Gaby

Thanks guys I've put a tonne of effort in so hope it pays off! I was going to fix up the references (cause they just have my websites without being real references) but it took me a lot longer than i thought for the images as you
can see its 5am. Nice. Yes i think linking the videos with the relevant parts is best. I have added links to videos throughout my section. Also I have made it so that when you put your mouse over the images it comes up with its own
reference/copy right info. Also when adding a link to another website its best to name the website instead of having just the web address. Its really easy if you don't know how and i can show you how to fix them up. When we get our
feedback from the other groups we can add and so forth before the project is actually marked which is really great. I think we need more info with the Genetics part but i figure bron is doing that tomorrow! Jo I really love your
sections pics and info it great and to the point!Gaby, yeah i did move it hope thats ok. I do have one question though and not meaning to hurt your feelings but i'm not that keen on the drawn image of the bird,fish,frog and mouse.
It doesn't really fit. Sorry, I mean obviously the decision is up to you, but i think it just looks a bit odd in the middle of the page because it you see it first and think , what? That was just some of the other feedback i got
from some of my friends that i asked to check out the website and see how they could maneuver around it. So that's my major spiel. Catch ya tomorrow. Sal