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There are many laboratory strains of mice that have Fertilization- union of male and female gametes
each been inbred to homozygosity at all loci. while avoiding hybridization and polyspermy
-each strain has a characteristic color Mechanisms have been studied on different on
animal models including sea urchins, however, few
-in experiments involving the embryo of
molecular mechanisms and features are common
more than one strain, the differences may serve as
with mammals, thus reducing value of invertebrate
a visual indication of genetic constitution.
models.
Products of mouse genes, like in humans but nor
Mouse sperm:
invertebrates, are spelled in capitals. (eg. NODAL)
-haploid
Mice mate at night thus, age of embryo is often
expressed as days and a half. -highly condensed DNA with protamines
(make up the protein content of chromatin)
-eg. If embryo is recovered on the eighth
day, it is designated as E7.5 (7.5 day embryo) -acrosome- Golgi body located infront of
nucleus
-solid white deposit or plug formed after
mating determines the mating night -behind the nucleus: centriole, midpiece
which is rich in mitochondria, tail having 9+2
Ovulation occurs few hours after mating and
arrangement of microtubules
fertilization takes place at the upperend of the
oviduct. -swimming movements are driven by dynein
arms attached to the microtubules
Numerical Stages by Theiler: (aside from the usual
“E” designations) Capacitation- (since sperm are not capable to
fertilize immediately after release) sperm spending
Stages 1-5 – preimplantation
a period of time inside the female reproductive
-embryos are located at the oviduct tract to become competent to fertilize.
Stages 6-14- early post-implantation -can also be brought out in vitro in media
containing albumin, calcium, and bicarbonate.
-contains body formation and turning
-involves loss of glycoproteins that prevent
Stages 15-27- organogenesis, fetal growth and birth serm-zona interaction, and display of acrosomal
(occurs 20 days after fertilization) protein at the surface.
3. increase intracellular Ca2+ and pH B 1,4 galactosyl transferase (GalT) cell surface
recognize the ZP3 of oocyte, and binding of ZP3
4. Increase motility
provokes acrosome reaction.
Mouse egg:
Acrosome reaction- rapid exocytosis of the
-Oocyte arrested MDII at metaphase. acrosomal vesicle. Materials released are:
-released from the ovary as complex with cumulus -hydrolytric enzymes like serine protease
cells
-acrosin- help digest a path through the
-surrounded by zona pellucida , a transparent layer zona and enables the sperm to reach the egg
of EC material secreted by follicle cells, embedded surface.
in hyaluronic acid
(for the ff see Fig. 10.3 on page 144 for the -spem contains 3 of this: fertilin a , fertilin B,
illutrsation of the mainsteps of sperm-egg and cyritestin
interaction)
Integrin-a6- best candidate for the target integrin
Hyaluronidase- assists the passage of sperm through on the egg because binding is prevented by a
the ECM of the oocyte-cumulus complex monoclonal antibody to this protein.
1. Injection of IP3 provokes Ca2+ release. In mammals they do not fuse to form a true
2. Inhibitors of phospholipase C or IP3 receptor zygote nucleus, instead the pronuclear
will inhibit Ca2+ release. envelopes breakdown as they meet, and the
3. Injection of whole sperm or chromosomes become aligned on the mitotic
demembranated sperm heads or sperm spindle ready for first cleavage.
extracts will provoke Ca2+ release.
4. Sperm contains specific phospholipase C In most mammals but not in mouse a centriole is
which cause release of Ca2+ contributed by the sperm and becomes the
5. Immunodepletion of phospholipase C from MTOC (microtubule organizing center) for the
sperm extract will demolish the activity. sperm aster, later dividing to form first mitotic
spindle.
Injection of Ca2+ or treatment of calcium
ionophore will cause same events of egg -In mouse, both centrioles are MATERNAL in
activation as fertilization by sperm. Such eggs origin, and this is the reason why
are parthenogenetic. (no paternal nucleus) parthenogenetic devt may occur.
-happens 3 days after fertilization where it (see figure 10.7 for illustration)
tranfers to uterus from the oviduct
Embryo hatches from zona and becomes
-cavity expands into blastocyst wich consists of implanted to uteri crypt
outer epithelial layer called tropoectoderm and
attached to its interior is the inner cell mass. -uterus is the only component to receive embryos
about 4 days from mating
- at 60-cell stage ¼ of cells are in ICM while
¾ are in tropoectoderm -mesometrial side of the uterus is the side
where the placentas form; implanted in such a way
ICM expresses a set of transcription factors that the ICM lies away from mesometrium
are pluripotent:
Trophoectoderm becomes trophoblast which
1. OCT4 (POU domain) stimulates the proliferation of uterine mucosa
2. SOX2 (SRY type) connective tissue to form deciduum.
3. NANOG (homeodomain)
4. FGF4 Nutrients from mother supply the embryo to make
the embryo grow and gain weight,
Tropoectoderm expresses:
Conceptus- entire product of fertilization
1. FGF receptor
2. FGFR2 Egg cylinder stage:
3. Transcription factors of TEAD4 (TEF family)
Cylinder is homologous to area pelucida of chick,
4. CDX2 (homeodomain)
consists of:
**3 and 4 are necessary for trophoectoderm
Primitive ectoderm or epiblast- “upper”
differentiation
layer
From E 3.5 to E. 4,5 both the ICM and
Primitive endoderm-“ lower” layer
trophoectoderm differentiate in two different tissue
types: **U shaped in sagittal section. O shaped in
transverse section**
Within the ICM:
Primitive endodermal cells (parietal endoderm)
-some cells lose expression of NANOG and
move to cover the inner surface of mural
acquire GATA4 and -6., these cells constitute the
trophoectoderm, anad start to secrete Reichert’s
primitive endoderm or hypoblast.
membrane, that contains laminin, entactin, and
type IV collagen.
Remainder primitive endoderm remains epithelial 2. Exocoelom separating amnion and
and forms the visceral endoderm. chorion
-Allantois is formed which grows
Distal region of inner egg cylinder: epiblast contact with chorion forming blood
vessels of placenta. (contains no
Proximal region: extraembryonic ectoderm derived
endodermal layer unlike in humans
from polar trophoectoderm.
and chick)
At E6.5: 3. Ectoplacental cavity lined with
extraembryonic ectoderm.
The anteroposterior region is apparent by -Placenta in contact with
formation of primitive streak (posterior end) mesometrium.
Definitive endoderm and mesoderm are Maternal placenta contains: 1) maternal decidual
formed by the extending of the streaks across tissues, which the trophoblast cells invades 2) giant
ectoderm towards distal tip of the cup. trophoblast cells with maternal blood sinuses
3)diploid trophoblast with fetal blood vessels 4)
Node: at anterior end of the streak (two cell layers
crypts lined with extraembryonic endoderm
while the remainder of streak has 3)
Placenta- nutrient supply for fetus; endocrine
At E7.5
functions like producing estrogen and
Headprocess is forming anterior to the node, progesterone.
with notochord that is flanked by:
At 8.5 days: TURNING (only in rodents; in humans
definitive endoderm in the lower epiblast are flat) happens, a process where germ
layer layers are brought to proper orientation within the
embryo by its rotation along its axis.
neural plate at the upper layer.
U shaped structure at dorsal side concave
Convergent extensions in forming trunk notochord. to U shaped structure at dorsal side convex.
At E8.5 Involves:
Chorion- farther from embryo; near ectoplacental Closure of midgut facing on the ventral side
cone becomes constricted to a small umbilical
tube which contains vitellointestinal duct,
Amnion-nearer embryo vitelline vessels, and allantois.
Amniotic fold divides promamniotic space into ORGANOGENESIS STAGES
three:
At E9.5, axis has formed, embryo has turned, and
1. Amniotic cavity above embryo gut has closed.
Mouse organogenesis is similar to chick. function)
1. Opticle vesicles.
Mouse organogenesis is similar to chick. E9.5 2. Neural crest emerges from neural
tube.
Neural tube closure takes place simultaneously with 3. Limb buds arise from
turning. Neural tube closure hindbrain then somatopleure.
proceeds anteriorly and posteriorly, 1. Posterior neuropore closing.
E10 2. Neural crest emerges from neural
Neural crest cells form skeletal structures of head, tube.
sympathetic NS, dorsal root ganglia, Schwann cells, 3. Genital ridges become visible.
pigment cells, adrenal medulla, and enteric 4. Germ cells enter hindgut.
5. Limb buds arise from
ganglia.
somatopleure.
1. Posterior neuropore closing.
65 somites at E14. Mostly found in the tail.
E10.5 2. Neural crest emerges from neural
tube.
Forelimb at 8-12th somite.
Germ cells starts to reach gonads.
Hindlimb at 23-28th somite. E11
1. Lens incorporated to eye. Left
Gut forms from fore and hindgut pockets. E11.5 and right atria become
separated.
Gut epithelium contributed by visceral and 2. Uteric bud is produced by nephric
definitive epithelium. duct.
Germ cells reach gonads.
5th pharyngeal arch is vestigial. E13
1. Somites continue to form until at
Mesoderm lateral to somites is the intermediate E14 this stage.
mesoderm which becomes the kidneys and 2. Hair follicles start to cover outer
epidermis.
gonads,
Activation FGF signaling upregulates Cdx2 A very rapid growth-> 660 to 15000 cells
(which is expressed by TEAD4) increasing the from egg cylinder to late streak stages.
proportion of trophoectodermal cells
produced. (ERK2 is involved in this process) NODAL plays a critical role in the sequence
of inductions:
CDX2- protein for trophoectoderrm
formation that suppresses Oct4 and Nanog. Nodal- expressed in the node;
Promotes original polarization event. expressed throughout epiblast and
downregulated in anterior and
At 64-cell stage two cell types have been concentrated at streak and node;
stabilized and no longer interconvertible. also needed for formation of anterior
visceral endoderm and primitive
After implantation ICM divides into outer primitive streak.
endoderm and inner core of epiblast.
Homozygous null embryos
FGF signaling triggers ICM cells to randomly are unable to form any
upregulate Gata6 and lose Nanog to sort embryonic pattern and
themselves into primitive endodermal layer genes for the formation of
at blastocoel surface the primitive streak and node
are not expressed
Primitive endoderm divides into:
The knockouts of the following abolish
1. Visceral endoderm- in contact with
anteroposterior polarity of embryo:
epiblast
2. Parietal endoderm- in contact with Activin receptors II A&B- receptors
mural trophoectoderm; for NODAL
-formed via delamination,
stimulated by fibronectin and Smad2- required for the signal
parathyroid hormone related transduction of NODAL-like factors
peptide and reduced by
laminin. Foxh1- partner of SMAD2 in
transcriptional regulation.
Trophoectoderm divides into two:
The anteroposterior pattern of the embryto arises
1. Polar trophoectoderm – near ICM from an egg cylinder that is initially radially
2. Mural trophoectoderm- surrounds whole symmetrical but polarized along the proxmodistal
blastocyst axis.
**division depends on proximity to ICM and requires Locations genes expressed in the egg cylinder
transcription factor ELF5 which is upregulated by (radially symmetrical):
FGF4 and NODAL**
1. Distal tip of visceral endoderm
EMBRYONIC BODY PLAN a. the transcription factor gene:
Hex
Tissue grafts within the epiblast develop in b. the inducing factor genes:
accordance with its surroundings. -Cerberus like 1(Cerl1)- CEBERUS
inhibits NODAL BMP and WNT
Regional determination is irreversible after late
action;
streak stage.
-dickkopf 1(Dkk1)- DKK1 protein
antagonistic to WNT action
-Lefty1 –LEFTY protein these prevents formation of forebrain but not trunk
antagonistic to NODAL action and posterior nervous system.
**Loss of Cerl1 and Lefty1 produces multiple FGF –CDX-HOX pathway controls patterning of the
primitive streaks** posterior region:
The establishment of the proximodistal pattern due FGF protein produces a gradient in all 3 germ
to the signals emitted by the extraembryonic layers:
ectoderm comprising WNT3 and BMP.
-arises by transcription of Fgf8 in the tail bud
Bmp2,-4, and 8bare expressed in the distal followed by decay of mRNA after the cells
extraembryonic ectoderm, knockouts of have left the tailbud.
these results in defective embryonic
mesoderm and primordial germ cells. Wnt genes are also important in posterior body
formation. (eg. Wnt3A)
As the cup shaped-egg cylinder expands and the
proamniotic cavity, the radial symmetry is broken. Retinaldehyde dehydrogenase- enzyme responsible
Morphogenetic movements shift the domains on for the production of endogenous retinoic acid; loss
diff’t side. of function lead to posterior defects.
Side containing the Hex domain- anterior Initial regionalization: head and trunk
Side containing the T domain- posterior Head pattern- depends on genes that are
upregulated in the anterior visceral
Distal visceral endoderm becomes the anterior endoderm, with inductive signal to form
visceral endoderm which expresses gene for anterior territory in epiblast.
secreted factors (including those mentioned earlier
at 1.b.) and genes for transcription factors (Otx2, Trunk pattern- depends on the signaling
Foxa2, and Lim=Lhx1) which are associated for center associated with the tip of the
anterior development. Knockout of these genes primitive streak and node (for neural
produces anterior truncations (shortening). induction and dorsalization of messoderm)
Denser mesenchyme due In the mouse, four hox genes clusters contains 39
transcription factors (PITX2 and ISL1) genes which are expressed at the phylotypic stage.
that produce N-cadherin.
-sharp anterior expressiom boundaries in the
One node cell contains a single cilium that is motile: central nervous system and mesoderm,
Fluid flow stimulates sensory cilia on the left -fades out posteriorly
side, provoking increase of intracellular
calcium which affects the gene expression. -2-3 per paralog group; per group have the
similar anterior boundaries
Three classes of mutants:
Knockout of hox genes results in an anterior
1. Randomization of Asymmetry transformation.
In the mouse, there are mutants that perturb -the gene distinguishes the overall hox
the normal left-right asymmetry due to cilia combination from that in more anterior positions.
defect.
-one hox gene knock out: modest effect
Normal= situs solitus
-one paralog group: substantial effect
Reverse= situs invsersus
K.O. of paralog 10 = lumbar covert
In mutants: expression of Nodal and Lefty2 to thoracic
may be on either side.
K.O. of paralog 11= sacral converts
Mutations are in genes encoding the to lumbar
motor proteins (needed for cilia
assembly (kinesin) and motion
(dynein) )
Substantial redundancy is because paralog 3. A large volume of mesenchyme
members are usually similar to each other and are appears between the primary
expressed in similar domains. endoderm and trophoblast