The Mouse Embryo
Written on 4:48 PM by gahrbeyj
Introduction
Model organisms are species biologists choose to study not only for inherent medical, agricultural or economic value but also for its inherent traits that make it easy and convenient to work with (The Gale Group, Inc., 2003). Most models have common characteristics that make them amenable for study. They are usually small, easy and inexpensive to breed. Most also have small genome sizes as well as sexual reproduction for cross-breeding. So far, we have tackled the fruit fly Drosophila melanogaster and the chick Gus gallusas model organisms. In this guide, we turn our attention to the once humble mouse, Mus musculus.
Mus musculus is the closest model organism to humans that is easy to manipulate and breed; bout 85% sequence similarity (The Gale Group, Inc., 2003). Its genome is about the same size as the human genome and the order of genes on the chromosomes is very similar in both organisms.
Genes are also more easily studied in mice. The mouse has become an invaluable genetic model system due to knockout genes technology. This generates mutant mouse strains where known genes have been deleted from the genome of every cell, thus allowing for gene function determination. This also serves to create models of study of genetic diseases. Indeed, The Jackson Laboratory has developed mouse disease models which are quite helpful in the understanding of these diseases. This includes down syndrome, cystic fibrosis, cancer,glaucoma, diabetes, epilepsy, heart disease and muscular dystrophy(Spencer, 2007).
The wide area of information about mouse genetics has made it a valuable model system for study of mammalian development as well as human genetic diseases. Its study is therefore important for understanding of development in higher mammals such as humans. In this experiment,the developmental changes mice embryos at ages 8.5-15.5 days post coitus undergo during fetal development are examined.
Methodology
Males and females are housed in the afternoon. Copulation can be confirmed by the presence of a vaginal plug. The time that the plug was found is considered 0.5 days post coitus assuming that copulation occurred at midnight the night before. Embryos are then obtained ranging from8.5 dpc to 15.5 dpc. These are rehydrated by placing in decreasing concentrations of methanol in PBS starting from 75% to 50% and thenin 25%. The embryos are then submerged in PBS solution and observed;small embryos are viewed under the light microscope and the larger embryos are viewed under the stereomicroscope in the Laboratory of Cell and Molecular Biology (LMCB).
Stages
In this experiment, the embryo stages observed were only from 8.5 to15.5 dpc. Below is a summary of the stages prior to 8.5 dpc.
Visit EMAP for Criteria Summary Table
8.5days post coitus. 8-12 somites
Turning:
Image fromTheiler 1989
The embryo at the 8 ½ day per coitum starts turning. It results in a change in the curvature of the embryo. The embryo starts from a lordotic curvature wherein there is a concavity at the anterior of the curvature of the lumbar and cervical spine to a dorsal kyphotic bend wherein there is a strong curvature of the upper back (Theiler1989 ; Farlex, 2008). The turning is initiated in embryos with6-8 pairs of somites. It is started to the head and tail folds and proceeds clockwise along the body axis as viewed from the cranial toward the caudal end.
Organogenesis
There is not much change occurring at this stage. Evident are the following organ rudiments: notochord, optic vesicles, trunk somites, branchial arches and gut.The optic evagination is deeper and the otic plate is more distinct(Theiler,1989).By the end of this stage, the 1stbranchial arch has its maxillary and mandibular components and a 2ndbranchial arch is evident. Regionalization of the heart is also discernible where a primitive ventricle, an atrium and a sinus venosus can be seen. Arterial and venous systems are also evident.
9 days post coitus. 13-20 somites
External Form
The embryo turning is complete and the embryos are flexed in a dorsally convex direction. There is also spiral torsion where the posterior end is usually found lying on the right side of the head.
This phase is most distinguished by the shape of the neural tube: the medullary plate is about to close at the anterior while there is still a flat groove at the posterior. The first 2 branchial arches are now very evident. There is also an increasingly prominent ridge at the lateral body wall indicating the site
of the future forelimb bud (Theiler, 1989, The University of Edinburgh, 2003).
The overall length varies due to the varying curvature of the body. From the crown to the curved posterior end, it has a length of 1.2-2.5 mm when straightened.
Circulatory System
The close-meshed capillary network investing the neural tube drains into the anterior cardinal vein which joins the posterior cardinal vein forming the Ductus Cuvieri. The paired dorsal aorta is seen ventral to the somites.
The heart is already capable of a little circulation of blood although the atrium and ventricle are not yet paired (Theiler, 1989).
Central Nervous System
The defining feature of this phase is the closure of the anterior neuropore which, interestingly, is accomplished at the same stage as humans.
At this stage, the eye anlage is in the vesicular stage. The optic evagination reaches overlying epidermis inducing the formation of the lens placode recognizable through tall epithelial cells formed after the disappearance of the anterior neuropore (Theiler, 1989).The olfactory placode meanwhile appears as a thickening in the epithelium. It is at first adjacent to the forebrain and becomes separated by invading mesenchyme.
The otic plate at this stage is transforming into a deep open groove. The invagination proceeds increasingly with somite numbers.
9.5 days post coitus . 21-29 somites
External Form
At this stage, the most prominent feature is the forelimb bud which first becomes apparent, usually found at the 8th-12thsomite. All three branchial arches are now visible at this stage.
Theoptic vesicle has not yet invaginated while the otic vesicle is still closed. The anterior neuropore is already closed after the 9thdpc. The length ranges from 1.8-3.3 mm in the fresh state (Theilre,1989r).
Circulatory System
The heart of the embryo is still an undivided tube. Endocardial tubes maybe recognized through the myocardial wall which is transparent (seeFigure).
The first aortich arch is still very small as compared to the 2ndand 3rd arches which are already well developed. These serve to conduct the blood through the paired dorsal aorta and through the umbilical artery which leads into the placenta.
The sinus venosus is still evident and still drains the same tributaries but already separates from the atrium though a transverse ridge.
Central Nervous System
At this stage, the posterior neuropore starts to close; the beginnings of constriction are evident. The brain also starts to develop rapidly. Optic vesicles have large stalks. At the outer periphery,cells of optic neural crest spread into the surrounding mesenchyme.
The lens placode and olfactory placode also start to appear as thickening of the surface epithelium.
The ear vesicle or otocyst detaches completely from the surface. The otocyst’s epithelium is highly specialized and different from simple skin ectoderm. It has tall cells and mitosis occurs only at the superficial layer bordering the lumen. This makes it resemble the wall of the neural tube.
10 days post coitus , 30-34 somites
External Form
Prominent in this features is the hind limb bud which first becomes apparent. It is seen as a bulge at the 23rd-28th somite. The precursor of the tail is also visible as a short stump. The concavity of the 3rd and 4th branchial arch is evident as compared to the 2nd. This forms the cervical sinus. The lens plate at this stage is also slightly indented while the olfactory placode is clearly indented. The otocys is quite closed and becomes pear-shaped in contrast to its previous spherical form. The posterior neuropore also starts to close,
At this phase, the length of the embryo varies between 3.3 and 3.9 mm(Theiler, 1989).
Circulatory System
At this phase, the cardinal veins may be distinguished. The heart is discerned as a prominent bulge near the branchial bars.
Central nervous system
The neural tube at this stage is completely closed. The otic vesicle is also completely closed and separated from the epidermis.
The olfactory placode is thicker and a little indented. The ganglia meanwhile appear as blastemal condensations (Theiler, 1989).The trigeminal ganglion can be seen situated anterior to the pointine flexture of the brain tube.
It may also be possible that the lens plate is indented at this stage.
10.5dpc (no details needed)
The distinguishing feature of this phase is the deep lens indentation where the lens pit deepens. It is accompanied by the narrowing of a pore-like opening and the appearance of the umbilical hernia. The 1stbranchical arch is now conspicuously divided into maxilliary and mandibular parts. The brain tube develops and there is tail thinning and elongation (The University of Edinburgh, 2003)
The distinguishing feature of this stage is the closure of the lens vesicle. Somites at the cervical region are not visible anymore while brain growth is very evident. Nasal pits start to form at this phase(The University of Edinburgh, 2003).
Lens vesicle completely separated from surface
45-47somites
6-7mm
External Form
During this stage of the mouse embryo, the paddle-shaped 'handplate' (anterior footplate) has formed in the anterior limb bud. The posterior limb buds are not yet divided into leg and foot. The nostrils are visible as narrow slits known as nasal pits. The medial and lateral margins of the otic pit are approaching each other,reducing the entrance to a narrow slit and the auditory hillocks become visible. The posterior somites are sharply defined and the tail is significantly longer than the previous stage. There is still no sign of retinal pigmentation and formation of digits.
Circulatory System
The aortic arch complex has become mature. The asymmetry of the umbilical veins becomes highly noticeable. The atrium is almost completely separated by the seprum primum. The ventricle (ventriculus communis)is still not separated into two chambers.
Intestinal Tract
The elevation of the tongue is not yet noticeable. The thyroid primordium grows deeper and loses its lumen. The primary bronchi start to develop secondary branches (lobar bronchi). The pharyngeal pouches starts to specialize into thymus and parathyroid but they are not yet completely separated from the pharyngeal epithelium. The stomach is enlarged and is separated from the outgrowing pancreas. The liver is composed of broad hepatic cords separated by large sinusoids. The cecum is noticeable as a short swelling of the colon and marks the boundary between the colon and the small intestine. The hindgut continues as the tail gut posteriorly and ends in the blastema of the tail tip.
Urogenital System
The genital ridge, which is still in its undifferentiated stage, contains numerous goncytes, which by then have completed their migration. The Wolffian ducts terminate at the cloaca. The ureteric buds are generally inflated and surrounded by condensed metanophrogenic tissue. The mesonephros is more mature and the mesonephric tubules are more apparent.
Central Nervous System
The most distinct feature of this stage of the embryo is that the lens vesicle is completely enclosed and detached from the ectoderm. The pigment layer of the optic cup has differentiated cells which contain pigment granules. The endolymphatic duct is longer. The nasal pit is separated from the oral cavity by the bucco-nasal membrane.
The ganglia of the cranial nerves have developed significantly. The anlage of the sympathetic trunk is apparent. There is not much significant brain development from the previous stage.
Numerous superficial folds that are well-vascularized have formed in the yolksac.
Earliest signs of fingers
48-51somites
7-9mm
External form
A distinguishing characteristic of the stage of the embryo are the changes that occur in the extremities. The 'handplate' (anterior footplate) is no longer paddle-like or circular. The developing angles visible are the early manifestations of finger rays. The posterior footplate is apparent and becomes distinguishable from the lower part of the leg. The somites are highly noticeable from mid-trunk to the tail. The spinal ganglia may be visible through the skin. Retinal pigmentation is apparent. Tongue becomes well-defined. Brain vesicle is observable.
Anterior footplate indented; Marked pinna
52-55somites
9-11mm
External Form
The rapid development of the pinna is highly noticeable and forms a crest at right angles to the head. Five rows of whiskers are visible and prominent hair follicles form above the eye and in front of the ear. Another distinguishing characteristic of this stage is the indention of the 'handplate', making a distinction between the elbow and the wrist. The skeleton of the forelimb now contains cartilage but the 'hand' remains mesenchymous. The footplate is just starting to become indented. Somites are clearly visible at the distal part of the tail,which is usually curved to the left rather than the right. The choroid plexus in the brain extends into the lateral and fourth ventricle, forming finger-like evaginations.
Circulatory System
Boththe aortic and pulmonary trucks have completely separated. All valves of the heart are already present in their primitive form but circulation is not yet continuous at this stage.
Intestinal Tract
There oral cavity maintains connection with the nasal cavity. The dental lamina of the future molars is already visible. The epiglottis is restricted by a discrete cleft from the rest of the larynx, which further projects into the pharynx. The lungs become highly lobed and the segmental bronchi continue to branch. The thymus completely separates from the pharynx. The thyroid is still a solid complex. The liver at this stage is already well-developed, with scattered blood-forming foci. The stomach still lack differentiated glands. The spleen appears as a triangular structure. The pancreas produces numerous sprouts that swell the dorsal mesentery.
Urogenital Tract
During this stage there is rapid development of the kidneys and sexual differentiation. The mesonephros already contains many regressing mesonephric tubules. The ureter is continuous with the swollen pelvis. The ureter comes in contact with both the Wolffian duct and the urogenital sinus. The Wolffian duct is more developed in the male while the Mullerian duct in the female.
The sex of the gonads may already be identified. Male primordial germ cells are located centrally while female germ cells are peripherally located. Some of the female gonocytes start to divide.
Central Nervous System
Prominent changes occur in the deiencephalic roof. The choroid plexus is already fully developed. From a small evagination during the previous stages, the pineal gland is now highly recognizable. The hypophysis develops rapidly. The lens vesicle becomes spherical as it loses its lumen. The layer of nerve fibers in the retina form a wide border with ends tapering peripherally. The clear nuclei of future ganglionic cells are already apparent in some regions.
Stage22 - 14 dpc
Fingers separate distally
56-60somites
11-12mm
External Form
A distinct characteristic of this stage is the separation of individual fingers in the anterior footplate. The posterior footplate show deep indentations between developing toes but separation is not yet observable. Long bones of the limbs are clearly visible. Hair follicles may be observed in the skin but not in the head region. The developing pinna turns forward and covers about half of the external auditory meatus. The umbilical hernia is highly striking at this stage.
Toes separate
60-65somites
12-14mm
External Form
It is during this stage of the embryo that and toes and finger are fully-formed. The pinna covers more than half of the external auditory meatus. The superficial veins are highly visible. Hair follicles are visible all over the body. The eyelids are open. The somites are no longer detected by external examination.
Circulatory System
The atrio-ventricular and semilunar valves of the heart are fully developed. The wall of the ventricles is smooth on the external side and has indentations of small sinuses on the internal side. The arteries and veins are already in their final fetal configuration and provide a bigger umbilical circulation than the yolk-sac.
Intestinal Tract
The oral and nasal cavities are completely separated. The salivary glands form distinct glandular branches. The cartilage of the larynx and upper trachea are now well-developed. The lung tissue is still compact but is more intensely vascularized than the previous stage. The thyroid develops into small buds abundant of blood vessels. The parathyroid becomes embedded in the thyroid. The thymus can be recognized as a distinct lymphatic organ with many free lymphocytes in the center. Numerous thick villi have developed in the small intestine while crypts have formed in the large intestine. The stomach becomes divided into 2 parts, the glandular area with its columnar epithelium and the nonglandular area with its cuboidal and stratified epithelium. The spleen now contains numerous blood vessels. The umbilical bernia still exists.
Urogenital Tract
The capsule of the kidney is bordered by a wide layer of blastermal tissue. The kidneys show large glomeruli at the central region.
Sexual differentiation has greatly advanced. The ovary contains a great number of gonocytes that are often clustered. The seminiferous tubules of the testis are already well differentiated. Sex can easily and accurately be determines
Central Nervous System
Cortex of the developing hemisphere is still visible during this stage. A small cellular area, which other consider as the paraphysis, form at the anterior boundary of the diencephalic roof.
REFERENCES:
Baldock R, Bard J, Davidson D and Lawson K. (25 October 2002). The Edinburgh Mouse Atlas: Staging Criteria. Retrieved 20 July 2008, fromhttp://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.shtml
"ModelOrganism." Genetics.The Gale Group, Inc, 2003. Answers.com20Jul. 2008. http://www.answers.com/topic/model-organism
Spencer,Geoff. 2007. Background on Mouse as a Model Organism in NationalHuman Genome Research Institute. Retrieved July 20, 2008 inhttp://www.genome.gov/10005834
Farlex.2008. Kyphosis in TheFree Dictionary: Medical Dictionary. Retrieved July 20, 2008 fromhttp://medical-dictionary.thefreedictionary.com/kyphotic
Theiler,Karl. 1989. The House Mouse: Atlas of Embryonic Development. NY:Springer-Verlag
The University of Edinburgh. 2003. The Edinburgh Mouse Atlas Project inThe University of Edinburgh Medical Research Council.Retrieved July 20, 2008 fromhttp://genex.hgu.mrc.ac.uk/intro.html
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