2009 Group Project 3: Difference between revisions

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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).  
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]]


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. http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F2.html  
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. http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F2.html  
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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).  
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:Lightbulb.jpg|center]]
[[File:Lightbulb.jpg|center]]

Revision as of 14:26, 11 September 2009

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 particular unique characteristics which allow it to be more easily studied than others. These advantages include the high proliferation 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, allowing for the 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)


This is different to the study of other embryos in that their development must cease 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.

History of Model Use

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,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.

Charles Kimmel

Professor 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).

1981 remake of Nature journal.jpg

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. http://www.nature.com/nrg/journal/v3/n9/fig_tab/nrg892_F2.html

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).


Lightbulb.jpg


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.

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. 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 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 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.

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.

Stages and Timeline of Embryonic Development

The Embryonic development of the Zebrafish can be broken down into

Images from here http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_stage.html


Zygote Period

The duration of this period is from 0 to 0.75 hours

  • It is here that the 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 stages


Cleavage Period


The duration of this period is from 0.75 to 2.25 hours

  • The transition during this period is from two cells to 128 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’.


Blastula Period


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 Midblastula Transition (MBT) where the yolk layer forms 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


Gastrula Period

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


Segmentation period


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 elogates and the tail bud (at the caudal end) becomes more prominant
  • The first body movement occurs
  • The Kupffer vessle in the Tail can be seen at its base


Pharyngula Period

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
  • Nervous system is hollow and expanding anteriorly
  • The brain has developed into 5 distinct lobes
  • Seven pharyngeal arch's develop rapidly during this stage
  • Pectorial 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


Short video of a Zebrafish Twitching: http://www.youtube.com/watch?v=0hGT667ktTw

Video of a Zebrafish embryo heart beating: http://www.youtube.com/watch?v=5ygcu9BRXI0


Hatching Period

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
  • Pectorial fins are now elongated with a flat blade but still developing
  • Jaws and Gills are also developing along side the pectorial 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
  • Subintestinal vein is also prominant ventrally to the gut tract formation


Larval Period

The Duration of this period is from 72hours to 30days

  • The Transition from Protruding-mouth to Day 30-44
  • The pectoral fin continues to develop and the internal organs become more complex
  • Inflation of the swim bladder


Juvenile Period

The duration of this period is from 30-44 days

  • Here adult fins and pigments as well as 12 teeth develop.
  • The reflective strips of iridophores brighten and lengthen along the dorsal perspective
  • The gut drops to become more ventral


Adult Period

The duration of this period is from 90days to 2 years

  • Full Breeding Adult.


Videos


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

Movie- zebrafish egg developing over a 24 hour period

"Digital zebrafish embryo provides the first complete developmental blueprint of a vertebrate"(New Scientist magazine)


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

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 Stages of embryonic development of the zebrafish ; PMID: 8589427 [PubMed - indexed for MEDLINE

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 2, 2009, 1,540,405,905 base pairs have been sequenced of an estimated total of 2,011,659,089 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.

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.

Current Embryology Research

References

ANAT2341 group projects

Project 1 - Rabbit | Project 2 - Fly | Project 3 - Zebrafish | Group Project 4 - Mouse | Project 5 - Frog | Students Page | Animal Development