2009 Group Project 4
THE MOUSE (Mus musculus)
Introduction
The mouse is a small animal which can be effectively used as a model to study embryological development. It belongs to the class mammalian and order rodentia and shares a significant similarity of homology with humans. The mouse is one of the most commonly used animals as model organisms in experimental embryology and most sciences. The mouse is a useful mammalian model for human embryological development because the mouse;
- Has the same size genome as the human genome
- Genes can be easily manipulated and studied
- Have a high degree of homogeny with humans
- Produces a large offspring in a short amount of time
- Are not limited in ethical issues as they can be manipulated in ways which would be unethical to do to humans
- Are small organisms and are easily maintained
- Are not expensive
Reproduction
Mice are polyestrous meaning they can breed many times during the year and can also reproduce all year round. The male and females are capable of breeding at 50 days old and females begin their estrous cycle (go on 'heat') between 25-40 days old. The estrous cycle is 4-5 days in duration and ovulation occurs spontaneously. A mouse produces an average litter of about 10-12 mice and begins a normal estrous cycle 3-10 days following partuition. Having an understanding of mouse reproduction, can highlight the useful nature of studying the mouse as a mamallian model for human emryology.
History of the use of the Mouse Embryo Model
Introduction
The mouse has had a significant contribution to the field of Biomedicine since the middle of the sixteenth century (Hendrich et al. 2004). During the twentieth century, the use of the mouse embryo in particular increased significantly, and continues to be a popular experimental choice for researchers.
It's popularity over time in mammalian biology, biomedicine, immunology, oncology, pathology, and genetics is a result of:
- The efficient mouse breeding system: Which consistently monitors the characterisitcs, that are precisley known, generation after generation, thereby producing highly standardised strains (Hedrich et al. 2004).
- The metabolic and internal anatomical similarities between the mouse and the human, which allows for comparisons. Because of these similarities, they share similar diseases, for example, cancer, diabetes, autoimmunity, endocrine disease, and neurological dysfunctions (Hedrich et al. 2004).
- The ability to manipulate and 'shut-down' genes from the Mouse genome(???).
Please note that the following findings are based on research which have utilised the model of the mouse embryo.
Gregor Johann Mendel (1822-1884), Augustinian priest and scientist
In his notes One Hundred Years of Mouse Genetics: An Intellectual History II The Molecular Revolution (1981–2002), Kenneth Paigen wrote that Mendel's first experiment on the transmission of inheritance was made using mice. Due to complaints from members of the Catholic Church about the smell , he changed his material to Pea plants (Berry 1987).
1895: Robert Heinrich Johannes Sobotta (1869-1945), German Anatomist [1]
Researched the fertilisation and cleavage of the mouse's egg (Sobotta, 1895). Link to Dr. Sobotta's research [2]
What did he find?
- The corpus Luteum is formed by the enlargement of the epithelial cells of the follicle, aided by growth of connective tissue.
- There is no distinction between corpora lutea vera and corpora lutea spuria.
- The corpora lutea do not degenerate. They remain unchanged during the life of the animal, and therefore add to the size of the ovary.
1949: John H. Hammond, Jr. (1888-1965), Animal Husbandry Scientist
"First report of attempts to culture mouse embryos in vitro to the Blastocyst stage..." (???)
What did he do?
Cultured eight-cell morulae and four-cell-stage embryos to the Blastocyst sage.
Embryos removed at the two-cell stage soon after died. (???)
1956: Wesley Kingston Whitten (1918-), Australian Veterinary Scientist
What did he do?
Cultured 8-cell mouse embryos to the Blastocyst stage.
How did he do this?
Used a medium containing Krebs-Ringer's bicarbonate solution supplemented with glucose and bovine serum albumin (???).
Soon after, he found that some two-cell-stage embryos developed into blastocysts, upon some modifications to the original medium.
He moved to the United States later on, continuing his work in the well known, The Jackson Laboratory.
1958: Professor Dame Anne Laura Dorinthea McLaren (1927-2007), Developmental Biologist
Developed the first birth of mice in-vitro. Link to Published article [3]
1962: George Todaro (1937-) and Howard Green
- By studying the fibroblast cells of the mouse embryo , they explained how cells behave in a similar fashion once they had undergone transformation (Bhamrah et al. 2002).
How did they do this?
- Todaro and Green cultured the fibroblast cells from the mouse embryo.
What did they find?
- A few weeks after the culture, the growth rate of the Fibroblast cells slowed down. They thought that the cells, as with normal human fibroblast cells, would eventually stop didviding and die. However this was not the case.
- Two to three months later, the cell growth rate increased. This meant that the resulting new population of cells had undergone spontaneous transformation. The resulting permanently dividing cell line, called the '3T3 cell line', have been growing in culture for over 20 years. (Bhamrah et al. 2002)
1963: Ralph L. Brinster (1932-)[4], American Geneticist
What did he do?
- Determined the nutritional requirements of the pre-implantation mouse embryo.
- Established the microdrop technique that allowed two-cell-stage mouse embryos to be cultured to the Blastocyst stage (???).
1965: Beatrice Mintz (1921-)[5], American Embryologist
What did she do?
- Found that the zona pelucida of the mouse embryo could be digested using pronase (???).
- Generated adult chimeras. Link to description of a chimera [6]
1977: R.J. Mullen
What did he do?
Fused the normal and reeler mouse embryo at the morula stage to produce allophenic mice. ('The reeler mouse as a model of brain development')
1980: Ralph L. Brinster
What did he do?
The first to inject purified globin mRNA into mouse zygotes (???).
This formed the basis for the production of transgenic mice.
1983: Davor Solter (1939-), Developmental Biologist
What did he do?
Transferred nuclei between zygotes.
The importance?
Revealed the importance of parent gene imprinting in mammalian development (???).
1996: Toshio Ohshima, Jerrold M. Wardt, Chang-Goo Huht, Glenn Longenecker, Veeranna, Harish C.Pant, Roscoe O. Bradyt, Lee J. Martins, and Ashok B. Kulkarni
Generated Cyclin-dependant kinase 5 (Cdk5) null mice (a type of mutant mice) by homologous recombination to assess the role of Cdk5 in vivo. Targeted ES cells were injected into blastocysts to generate chimeric mice.
1998: Teruhiko Wakayama and Ryuzo Yanagimachi
What did they do?
Cloned the first mouse, named Cumulina.
1998: Nikola Skreb and colleagues
What did they do?
Showed that the early embryonic ectoderm contains cells capable of contributing to all three germ layers of the mouse fetus.
Use of teratocarcinomas
What are teratocarcinomas?
They are '...gonadal tumors that contain a...mixture of different tissue types, all derived from a population of undifferentiated stem cells known as embryonal carcinoma cells.' (???The white booklet)
The use of teratocarcinomas in research complement the studies on pluripotentiality of cells from the normal embryo and allows further study of early embryonic development.
First pioneered by Leroy Stevens (Jackson Laboratory), and Barry Pierce (University of Colorado)
Leroy C. Stevens, Jr.:
- The first to identify that male mice of the inbred 129 strain have a low incidence of testicular teratoma arising from primordial germ cells (Link to Stevens and C. C. Little's related article [7] PMID: 16578442).
- The first to identify modifier genes such as ter that increase the frequency of teratomas in the testis and eventually developed an inbred strain (129/Sv).
- Developed the LT strain, in which about 50% of females develop ovarian teratocarcinomas.
Ralph Brinster(1974)[8] PMID: 4610074 , Mintz and Illmensee (1975)[9] PMID: 1059147, and Papaioannou et al. (1975) [10] PMCID: PMC433040 demonstrated that embryonic carcinoma (EC) stem cells could be injected into blastocysts to create adult chimeras that contained normal tissues derived from the EC cells.
Timeline of Development
The development of the mouse embryo takes 19 days on average from fertilisation to newborn mouse.
Edinburgh Atlas Project[11]
- Development of the Mouse Embryo
Day 0 One Cell Egg
Day 1 Dividing Egg. Cleavage of cells has begun. The Day ends with 4 cells present.
Day 2 Morula
Day 3 Advanced segmentation of the Morula. 16-25 cells within same sized morula.
Day 4 Blastocyst formation
Day 4.5 Implantation and Invasion of the blastocyst into the uterine epithelium
Day 5 Formation of the egg cylinder
Day 6 Differentiation of the egg cylinder
Day 6.5 Gastrulation starts during the period of Advanced Endometrial Reaction
Day 7 Amnion formation
Day 7.5 Neural plate and presomite stage
Day 8 Formation of the first seven somites.
Day 8.5 Rotation of the embryo. The embryo now has between 8 and 12 somites.
Day 9 Formation and closure of the anterior neuropore. There are 13 - 20 somites present in the embryo
Day 9.5 Formation of the posterior neuropore and forelimb bud. There are 21 to 29 somites present and the embryo measures 1.8 - 3.3 mm in length.
Day 10 The posterior neuropore closes and formation of the hind limb bud and tail bud occurs. there are 30 - 34 somites present and the embryo is 3.1 - 3.9mm
Day 10.5 Deep eye lens indentation occurs. There are 35-30 somites present and the embryo is now 3.5-4.9mm.
Day 11 Closure of the lens vesicle. There are 40-44 somites present and the embryo has reached 5-6mm in length.
Day 11.5 The lens vesicle is completely separated from the surface epithelium. There are currently 45 - 47 somites and the embryo reaches a length of 6 - 7mm
Day 12 First signs of fingers. The embryo is 7 - 9mm in length from crown to rump and there are 48 - 51 somites.
Day 13 The anterior footplate becomes indented and the external ear structure is more defined. The embryo is now 9 - 11mm in length and has 52 - 55 somites.
Day 14 The fingers separate. The embryo has 56 - 66 somites and is 11-12mm in length.
Day 15 The toes separate. At this stage the embryo is 12 - 14.4m from crown to rump
Day 16 The umbilical hernia is repositioned. The fetus now measures between 14 and 17 mm.
Day 17 The fingers and toes become joined together. The skin is wrinkled and thickened and the umbilical hernia has disappeared. The fetus measures 16.5 - 20mm depending on the degree of curvature.
Day 18 Whiskers elongate. The length of the embryo varies between 18 and 23 mm due to the degree of curvature of the fetus.
Day 19 The newborn mouse is between 23 and 27mm depending on the degree of flexure of the body axis
Staging Of Embryonic Development
Theiler stages
Mouse embryonic development commences once the female's egg or oocyte has become fertilized by the male's sperm. Mouse development has a gestation period of 19-21 days, and can range in different strains of mice. The development of an embryo can be categorized into different stages including cell number, somite stages and morphology. The most common method of staging is by Theiler (1989) which categorizes mouse development into prenatal and postnatal stages consisting of 26 and 2 stages respectively. Downs and Davies (1993) have also established a method of staging the mouse development based on morphological changes. Link to Downs and Davies[12]
The developmental stages of the mouse embryo can be summarised in the tables and corresponding figures below in relation to characteristics at that stage. The first stage begins with fertiliation of the egg which divides into multiple cells to form a morulla and further go on to form a blastocyst which is ready for implantation.
| Theiler Stage | Embryonic Age in Days Post Coitum (dpc) | Stage Characteristic | Cell Characteristics | Zona Pellucida | Location | |
|---|---|---|---|---|---|---|
| 1 | 0-0.9
(range 0-0.25) |
One-celled embryo (fertilized) | One cell | Present | Ampulla | |
| 2 | 1
(range 1-2.5) |
Dividing egg | 2-4 cells
- 1st cleavage after 24hrs |
Present | Travelling down oviduct | |
| 3 | 2
(range 1-3.5) |
Morula (early to fully compacted) | 4-16 cells | Present | Oviduct (utero-tubal junction) | |
| 4 | 3
(range 2-4) |
Morula to Blastocyst
-Intra-cellular matrix -Blastocoelic cavity |
16-40 compacted cells
-Inner cell mass -Outer layer of trophectoderm cells |
Present | Uterine lumen | |
| 5 | 4
(range 3-5.5) |
Zona free Blastocyst (hatching) | Blastocyst implants as zona pelludica is lost | Absent | Uterine lumen |
Dr Mark Hill 2009, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
Once the Blastocyst has lost the zona pellucida, it is free to implant onto the uterine wall. After implantation multiple features of a trilaminar embryo start to develop. Ectoderm, endoderm and mesoderm layers form, which further develop many structures corresponding to different stages as summariesed in the table below. The developing mouse embryo which is implanted on the uterine wall can be viewed by ultrasonic radiation. Link to ultrasound of mouse embryo [13]
| Theiler Stage | Embryonic age in Days Post Coitum (dpc) | Stage Characteristic | Cell characteristics | |
|---|---|---|---|---|
| 6 | 4.5 (range 4-5.5)
Human carnegie stage: 4 |
Attachment of blastocyst
-Implantation |
Embryonic Endoderm | |
| 7 | 5 (range 4.5-6)
Human carnegie stage: 5 |
Implantation
-Egg cylinder -Ectoplacental cone |
Inner cell mass increases
-Epiblast -Mural trophectoderm lined by endoderm | |
| 8 | 6 (range 5-6.5)
Human carnegie stage: 5 |
embryonic and extra-embryonic regions
-Pro-amniotic cavity |
Trophoblast giant cells invade
-Maternal blood invades -Reichert's membrane | |
| 9 a) | Pre-streak | Advanced Endometrial and egg cylinder
-Embryonic axis |
Embryonic and extra-embryonic ectoderm
-Uterine crypts lose lumen | |
| 9 b) | Early streak | Gastrulation begins | Mesodermal cells | |
| 10 a) | 7 (range 6.5-7.5)
Mid to late streak Human carnegie stage: 8 |
Amnion formation | Amniotic fold
-Allantoic bud -Primitive node -Amnion closes | |
| 11 | 7.5 (range 7.25-8)
Human carnegie stage: 9 |
Neural plate and presomites | Amniotic cavity, exocoelom and ectoplacental cleft
-Allantoic bud elongates -Notochodal plate -Early head fold -Foregut pocket |
This table can be seen in more detail by following the link More on stage 6-11
The next stage (Theiler stage 12) is marked by the first somite pair formation. Stages 13 onwards is characterized by increasing somite pairs up to 20 somite pairs. These embryo stages have characteristic features of developing forgut, prominent head fold, optic pits, branchial arch and neuropore formation. These four stages can be summarised in the figures 4-7 below. These stages can be seen in table form by following the link Table of stages 12-14
Following stage 14, The embryo continues to develop more distinguished features of a mouse, including forelimb and hindlimb bud, tail elongation, lungs and brain development. The stages 15-20 are summarised in the table below.
| Theiler Stage | Embryonic Age (dpc) | Stage Characteristic | Cell Characteristic | Somite Number(pairs) | |
|---|---|---|---|---|---|
| 15 | 9.5 (range 9-10.25)
Human carnegie stage: 12 |
Forelimb bud (8-12th somite pair)
-Posterior neuropore |
Hind limb bud
-Telencephalic and diencephalic vesicle division -Lung development -Dorsal pancreatic bud |
21-29 | |
| 16 | 10 (range 9.5-10.75)
Human carnegie stage: 13-15 |
Caudal neuropore closes
-Hind limb and tail bud |
Concave 3rd and 4th branchial arches.
-Rathke's pouch -Nasal processes -Ventral pancreatic bud |
30-34 | |
| 17 | 10.5 (range 10-11.25)
Human carnegie stage: 13-15 |
Deep Lens Indentation | Physiological umbilical hernia
-1st branchial arch (maxillary and mandibular components) -Brain tube development -Tail elongates |
35-39 | |
| 18 | 11 (range 10.5-11.25)
Human carnegie stage: 13-15 |
Lens Vesicle closes | Cervical somites not visible
-Brain rapidly grows -Nasal pit |
40-44 | |
| 19 | 11.5 (range 11-12.25)
Human carnegie stage: 16 |
Lens vesicle separated
–Closed and detached from ectoderm |
Eyes and their peripheral margins
-Limb-girdle and arm -Anterior footplate. -Auditory hillocks |
45-47 | |
| 20 | 12 (range 11.5-13)
Human carnegie stage: 17 |
Gingers | Anterior footplate (develops angles)
-Posterior footplate -Pigmentation of retina -Tongue and brain vesicles |
48-51 |
This table can be seen in more detail by following the link More on stage 15-20 To visualize the mouse development so far a movie showing the mouse embryo can be seen by following the link [14]( note the heart beat)
The last few stages of develop (stages 21-26) shown in table 4 are the complete prenatal developmental stages. These stages involve fine development of the pinna over the external acoustic meatus, finger and toe,hair, eyes and whiskers.
| Theiler Stage | Embryonic Age (dpc) | Stage Characteristic | Cell Characteristic | Somite Number(pairs) | |
|---|---|---|---|---|---|
| 21 | 13 (range 12.5-14)
Human Carnegie Stage 18-19 |
Anterior footplate indented
-Pinna develops |
Digits, elbow and wrist
-5 rows of vibrissae visible -Hair follicle over eye and ear -Lens loses lumen |
52-55 | |
| 22 | 14 (range 13.5-15)
Human Carnegie stage 20-23 |
Individual fingers on anterior footplate
-Umbilical hernia visible |
Deep indentations between toes( not separated)
-Long bones of limbs present -Hair present in pectoral, trunk and pelvis -Pinna turned forwards |
56-60 | |
| 23 | Human fetal period | Toes separate | Hair follicles present in cephalic region
-Eyelids open -Pinna covering ½ of external auditory meatus |
>60 | |
| 24 | 16 | Reposition of umbilical hernia | Parallel fingers (2-5)
-Toe nail primordia -Eyelids fused -Complete coverage by pinna -Increase in peritoneal sac size |
>60 | |
| 25 | 17 | Skin thickened and wrinkled
-Umbilical hernia disappeared |
Subcutaneous veins less visible
-Fingers and toes parallel -Whiskers visible |
||
| 26 | 18 | Skin thickened
Long whiskers |
Pinna larger (auditory meatus lumen not visible) |
Following the prenatal development are the two Theiler stages 27 and 28 which involve birth of the mouse and postnatal development.
Genetics
Genome
Sequencing of the mouse genome was completed in late 2002. Tha haploid genome is about 3 billion long (3000 Mb distributed over 20 chromosomes) and therefore equal to the size of the human genome. The current estimated gene count is 23,786 and humans are estimated to have 23,686 genes.
The genetic map of the mouse
Genetic maps, the road maps of genetics, are of two types: linkage and physical. The 'sign posts' on the maps are loci, any location or marker in the genome that can be detected by genetic or DNA analysis. The term 'gene' is more restrictive than loci and refers to DNA segments that encode proteins or can be linked to phenotypes. Linkage maps are recombinational maps and are constructed by carrying out linkage crosses that measure the recombination frequency between genes or loci on the same chromosome.
- The first genetic linkage in the mouse (and first autosomal linkage in mammals) was described in 1915 in the classic paper on the linkage of pink-eyed dilution and albino (Haldane et al., 1915)
- Genetic mapping with spontaneous mutations that created visible phenotypes, such as changes in coat colour/texture or behaviour was labarious and sometimes took years because crosses between mice carrying recessive mutations yielded so few informative progeny, and genes on only one or two chromosomes could be scored in each cross.
- The first real breakthrough in linkage mapping, enabling the scoring of many test markers and chromosomes in the same cross, was the discovery and use of co-dominant biochemical (isoenzyme) genes (e.g. glucose phosphate isomerase 1, Gpil; Hutton and Coleman 1969).
How large is the genome?
With the use of Feulgen reagent the quantitative DNA-specific staining can be achieved. Through micro photometric measurements of the staining intensity in individual sperm nuclei, it is possible to determine the total amount of DNA present in the haploid mouse genome (Laird, 1971). Measurements indicated a total haploid genome content of 3 pg, which translates into a molecular weight of 1.8 x 1012 daltons (Da).
How complex is the genome?
Another method for determining genome size relies upon the kinetics of DNA renaturation as a sign of the total content of different DNA sequences in a sample. When a solution of double stranded DNA is denatured into single strands which are then allowed to renature, the time required for renaturation is directly proportional to the complexity of the DNA in the solution, if all other parameters are held constant.
Complexity is a measure of the information contained within the DNA. The maximal information possible in a solution of genomic DNA purified from one animal or tissue culture line is equivalent to the total number of base pairs present in the haploid genome.The information content of a DNA solution is independent of the actual amount or concentration of DNA present. DNA obtained from one million cells of a single animal or cell line contains no more information than the DNA present in one cell. Furthermore, if sequences within the haploid genome are duplicates of one another — repeated sequences — the complexity will drop accordingly.
Renaturation analysis of mouse DNA reveals an overall complexity of approximately 1.3-1.8 x 109 bp. This value is only 40-60% of the size of the complete haploid genome and it implies the existence of a large fraction of repeated sequences.
Comparative mapping
Comparative mapping began in the early 1970s and gained momentum until it culminated with the sequencing of the two genomes in 2001 and 2002.
- First conserved mouse and human autosomal linkage was reported in 1978.
- 13 conserved autosomal segments and estimated 178(+-)39 chromosomal rearrangements between mouse and human chromosomes were identified (Nadeau and Taylor,1984)
- Sequencing of the two genomes revealed that 95% of the coding sequence is conserved at the DNA level (Consortium,2002)
Current Research
References
1. Dr Mark Hill 2009, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G [15]
2. Bard, Kaufman, Dubreuil, Brune, Burger, Baldock, Davidson (1998). An internet accessible database of mouse development anatomy based on a systemic nomenclature, Mechanisms of development, (74) 111-120.
3. Theiler, K 1989. 'The House Mouse'. Springer- Verlag, New York
Begum's Reference (???)
1. Hedrich H., Bullock G., & Petrusz P. (2004). The Laboratory Mouse. London: Elsevier Academic Press.
2. Nagy A., Gertsenstein M., Vintersten K., & Behringer R. ( 2003). Manipulating the Mouse Embryo: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press.
3. Bhamrah H.S., & Juneja K. (2002). Molecular Cell Biology. 1st ed. New Delhi: Anmol Publications.
note: reference edinburgh atlas project
ANAT2341 group projects
Project 1 - Rabbit | Project 2 - Fly | Project 3 - Zebrafish | Group Project 4 - Mouse | Project 5 - Frog | Students Page | Animal Development
