- exocytosis of cortical granules, which contrain glycosidases and

THE MOUSE proteases that modify the zona pellucida receptors to prevent
- Viviparous = bringing forth live young that have developed inside the polyspermy
body of the parent 8. Nuclear decondensation
- inaccessibility of the postimplantation staged of development = - assisted by reduction of protamine disulfide bonds by peptide
microsurgical procedures are used less glutathione present in egg
- have considerable external nutrient supply and undergo extensive 9. Pronuclear fusion
growth - pronuclear envelopes break down as they meet and
chromosomes align on the mitotic spindle for the first cleavage.
MAMMALIAN FERTILIZATION
- the mechanism needs to bring the male and female gametes Preimplantation Stage
together in productive union while avoiding hybridization and  comprises the initial stages of mammalian development,
polyspermy. before the embryo implants into the mother’s uterus.
 2-cell stage- expression of zygotic genome commences
The Sperm  1st cleavage- 24 hrs
- Haploid, DNA is higly condensed with protamines (very basic  2nd & 3rd cleavage- 12 hrs
proteins)  slow development- adaptation to time required to prepare
- Acrosome in front of nucleus uterus for implantation
- Centriole behind nucleus  8- cell stage- compaction occurs
- Flagellum for swimming  Compaction
- Swimming movements are drivens in ATP-dependent o flattening of blastomere to maximize
process by dynein arms o facilitated by E-Cam
 cells become polarized
The Egg  Gap Junctions form
- Oocyte arrested in 2nd meiotic metaphase o Allow diffusion of low molecular weight substances
- Released from ovary as a complex with ovarian follicle cells throughout embryo.
- Surrounded by zona pellucida  Morula
- Complex is picked up by the infundibulum, “churned” to  Up until 32-cell stage
compress matrix and allowed through the ostium into the  Tight Junction, Desmosomes form
oviduct o Creates permeability seal between inside and
- Ovulation occurs a few hours after mating outside of embryo
- Fertilization takes place in the oviduct  Blastocoel forms
o 3 days after fertilization
9 events for a successful fertilization: o time that embryo moves from oviduct to uterus
1. Capacitation  Blastocyst
- process wherein the sperms need to spend a period in the  Blastocoel expands
female reproductive tract during which they become competent  Trophectoderm
for fertilization o Outer cell layer of epithelial morphology
- in vitro = simple synthetic media + albumin + calcium + o Transcription Factors for Trophectoderm
bicarbonate differentiation
- loss of cholesterol = makes membrane permeable to Ca2+ and  TEAD4
HCO3− > activate adenylyl cyclase > production of cAMP and  CDX2
activation of protein kinase A o Differentiates into
2. Secretion of hyaluronidase for penetration  Mural Trophectoderm- transformed to
3. Binding of sperm to zona pellucida polytene giant cells
Zona  Polar Trophectoderm- continues to
- composed of glycoproteins ZP1, ZP2, & ZP3 proliferate
- ZP3 = specific sperm receptor  Inner Cell Mass (ICM)
= low concentration will prevent sperm-zona binding o Gives rise to definitive structures of the fetus
- species-specificity resides in the carbohydrate attached to the o Transcription factors that maintains pluripotency
ZP3 polypeptide  OCT4
- recognition protein for ZP3 on sperm is β-1,4-galactosyl  SOX2
transferase (GalT)  NANOG
4. Acrosome reaction o Some cells lose NANOG
- rapid exocytosis of acrosomal vesicle  Replaced by GATA4 and GATA6
- due to binding of ZP3 to β-1,4-galactosyl transferase (GalT)  For cardiac development
- G protein activation > less negative membrane potential >
opening of voltage-gated Ca2+ channels > rise in intracellular Early Postimplantation Stages
Ca2+ and pH  Zona hatches
5. Digestion of zona  When embryos implant
- acrosome reaction released hydrolytic enzymes such as serine o ICM lies away from mesometrium
protease and acrosin  Trophectoderm becomes Trophoblast
6. Sperm-egg membrane recognition  Trophoblast
- carried out by ADAM (a disintegrin and metalloprotease o Stimulates formation of Deciduum
domain) that binds to integrins on the egg surface  Forms maternal part of placenta
- sperm has 3 ADAM proteins: fertilin α, fertilin β, and cyritestin  Egg Cylinder
7. Plasma membrane fusion o Homologous to area pellucida
- requires a four-pass membrane protein: tetraspanin or CD9, o Cup shaped
and one or more glycosylphosphatidylinositol (GPI)-anchored o It has
cell surface proteins on the egg  Epiblast- homologous to chick epiblast
- fusion causes the rise of intracellular Ca2+  Primitive endoderm
- Ca2+ release is caused by activation of inositol triphosphate  Move out to cover mural
(IP3) pathway by a specific phospholipase C introduced by the trophectoderm
sperm
 o Now called parietal  notochord
endoderm  neural plate
 Around epiblast  gene: Otx2
o Forms Visceral endoderm o 8.5 days
 Extraembryonic ectoderm  neural tube
 Derived from polar trophectoderm  somites
 Ectoplacental cone  1 somite per 1.5 hrs
 From polar trophectoderm  Turning (rodents only)
 forms trophoblast giant cells as it  Brings germ layer in their proper
proliferates orientation
o At about 6.5 days Human Early Development
 Anteroposterior axis- apparent due to
primitive streak  Takes 4 Days to move to Uterus
 Primitive streak  1st Division- 30 hrs
 Marks posterior end  2nd Division- 40 hrs
 Laterally compressed  Mature blastocyst 4th day
 Definitive endoderm and mesoderm  Also has trophectoderm w/c is called trophoblast
(Transcription factor gene: brachyury)  Day 7- primitive endoderm visible
(established)  Day 8- difference between mouse and human occurs
 Node appears o Amniotic cavity in ICM
o 7 Days o No egg cylinder formed
 Amniotic fold  Instead, hypoblast= makes blastodisc

o 7.5 Days
 head process is formed

Organogenesis Stage

E8.5 Fusion of paired heart primordia

E8.5-10.5 Emergence of neural crest from neural tube
E9 Closing of Anterior neuropore (Neural tube)
Heartbeat begins
Formation of the mouth & 6 pharyngeal arches
Rise of pronephric primordium (no function)
E9.5 Formation of optic vesicles
E9.5-10 Limb buds arise from somatopleure
E10 Genital ridges become visible
Germ cells enter the hindgut, migrate up the mesentery (E11-E13)
E10-10.5 Closing of posterior Neuropore (Neural tube)
E11.5 Separation of left and right atria
Lens become incorporated into the eye
Nephric duct produces the ureteric bud
E14 End of somite formation
E14- Hair follicles arise

Neural tube closing is simultaneous with turning

o Takes place in hindbrain
o Proceeds anteriorly and posteriorly
 Somites form until E14, by which time they are 65 (many are in the tail, which is longer than the chick’s)
 Somites form the vertebrae and myotomes; also contributes to the dermis
 Gut originates from fore- and hindgut pockets
 Intermediate mesoderm becomes the kidney and gonads
 Lateral part of genital ridges becomes mesonephros
 Medial part of urogenital ridges produces the gonads
 Germ cells originate from extraembryonic mesoderm
 Lateral plate (it is lateral to the intermediate mesoderm) is divided by the coelom into:
o Outer somatopleure (epidermis and somatic mesoderm)
o Inner splanchnopleure (endoderm and splanchnic mesoderm)
 Limb buds:
o Forelimb bud – 8-12 somites
o Hindlimb buds -23-28 somites
Fate Map

 Fertilized egg does possess a polarity, which is retained through the early embryonic stages
 The original animal pole of the egg can be identified by the position of the 2nd polar body
 1st cleavage  meridional; separates blastomeres that will preferentially become the embryonic pole and the abembryonic pole of the
blastocyst
 Trophectoderm arises from the polar cells on the exterior of the morula
 Inner cell mass arises from the apolar cells in the interior
 There is a relationship between the axes of the blastocycst and the later egg cylinder stage:
o Cells on Animal pole  End up distally in the visceral endoderm of the egg cylinder
o Cells on Vegetal pole  proximally in the egg cylinder
 Primitive streak and mesoderm of the amnion and allantois all arise from the posterior edge of the epiblast
o Anterior part  becomes the node  notochord and part of the somites
o Middle part  Populates mainly lateral plate mesoderm in the posterior half
o Posterior part  populates mainly mesoderm of the amnion, visceral yolk sac, and allantois

Regional Specification end in epiblast

Formation of embryonic structures
 The early blastomeres are known to be totipotent
 From the 8-cell stage, it is no longer possible to obtain a
complete embryo from one blastomere
 The formation of ICM and trophectoderm depends on cell
polarization at the 8-cell stage = PAR proteins are involved:
EMK1 (=PAR1) - internal
PAR6b – external
 Administration of dsRNA will increase proportion of cells
becoming ICM and reduce trophectoderm
 ERK2 (concentrated apically) is involved in FGF signaling
 Activation of FGF signaling upregulates Cdx2 and trophectoderm
 TEAD4 expresses Cdx2 and CDX2 for trophectoderm formation
and suppresses expression of Oct4 and Nanog in trophectoderm
 After implantation, the ICM becomes divided to outer layer of
primitive endoderm and inner core of epiblast
 The trophectoderm becomes divided to polar trophectoderm and
mural trophectoderm
 The maintenance of cell division requires the transcription factor
ELF5 whose expression is upregulated by FGF4 and NODAL
 The primitive endoderm becomes divided into visceral endoderm
and parietal endoderm
 The formation of parietal endoderm is stimulated by fibronectin
and parathyroid hormone related peptide (PTHrP) and reduced
by laminin
 The establishment of proximodistal pattern is due to signals
emitted from extraembryonic ectoderm comprising WNT3 and
various BMPs
 Knockouts of these genes are early lethals, Wnt 3 (no primitive
streak or mesoderm) and individual Bmp (defective in embryonic
mesoderm and primordial germ cells)
 Morphogenetic movements shift the Hex domain (anterior) to
one side and T domain (posterior) to other
 The former distal visceral endoderm becomes the anterior
visceral endoderm (AVE) and expresses a group of genes for
transcription factors (including Otx2, Fox2a and Lim =Lhx1)
and genes for secreted factors (including Cerl, Dkk1 and Lefty
1) that are associated with anterior development
 The posterior epiblast, then the anterior streak then the node
itself all express genes associated with the organizer such as
Foxa2, Lim1 and goosecoid (Gsc)
 Chordin and Noggin are both expressed in the anterior streak
and node epiblast and these proteins will themselves induce
anterior neural tissue
 The FGF-CDX-HOX pathway controls the patterning of the
posterior region
 Wnt genes particularly Wnt3A is expressed in the posterior
 In all vertebrate species, the NODAL type factors have a key role
in mesoderm formation, BMP inhibitors are required to some
extent in neural induction and FGF and Wnt systems are
important for formation of lower parts

Embryonic body plan Germ Cell Formation

 The knockout of activin receptor IIA and B (Acvr2a and Acvrb) or -Primordial Germ Cells (PGCs)
of Smad2 or Foxh1 all abolish the anteroposterior polarity of the  Do not arise from a cytoplasmic determinant
embryo  Formed by induction
 The formation of primitive streak requires an epithelial-  30-40 are visible at E7.5 located at the base of the
mesenchymal transition allantois
 The transcription factor gene Hex and the inducing factor genes  induced by BMP4 from the extraembryonic
cerebrus like-1 (Cerl 1), dickkopf 1 (Dkk1) and Lefty1 are all ectoderm
expressed at the distal tip of visceral endoderm  PGCs reactivate expression of Oct4 and Sox2
 A number of genes including T are expressed at the proximal
Left-Right Symmetry
 Nodal becomes expressed preferentially on the left side of
the node from the two-three somite stage, along with Lefty2
 Expression spreads to the left lateral plate mesoderm
 Ex. 2 bHLH transcription factors contribute to the asymmetry
of the early heart:
o Cascade by NODAL -> causes dHAND to be
expressed on the right while eHAND on the left
 Counterclockwise coil of intestine:
o Presence of N-cadherin on the left, whose
synthesis is controlled by PITX2 ans ISL1
 Situs
o Original cause of asymmetry
o Situs solitus – normal
o Situs inversus
 3 distinct classes of mutant phenotype:
o mutants that cause randomization of asymmetry
 e.g. Nodal and Lefty2 on right side
instead of left: present in motor proteins
o mutant that leaves the embryo in a state of
bilateral symmetry
 e.g. TG373 mutant ->causes loss of
function: no cilia and symmetrical
expression of Nodal
o mutant has a reversed symmetry (situs inversus)
 e.g. inv
Hox Genes
 4 Hox gene clusters contain 39 genes
 paralog groups have 2 or 3 members
 Phylotypic stage
o Genes are maximally expressed in this stage
o Vertebrates tend to have sharp anterior expression
boundaries in the CNS and mesoderm and to fade
out in the posterior, and members of the same
paralog group have similar anterior boundaries
o Knockout
 Anterior transformation
 E.g. Knockout of Paralog group 10 =
Lumbar ->saral
 Knockout of paralog group 11 = sacral-
>lumbar
o Ectopic expression
 Posterior transformation
 E.g. Hoxa7, normally expressed with its
anterior boundary in the thoracic region,
is expressed in the head, then the basal
occipital bone of the skull is transformed
into a pro-atlas type vertebra.
HUMAN EARLY DEVELOPMENT
 Placenta begins to form with the appearance of lacunae in
the syncytuitrophoblast
o These become continuous with maternal blood
sinusoids
 Chorionic villi  projections of blood vessels derived from
the extraembryonic mesoderm
o Grow into blood-filled lacunae  provide for an
exchange between maternal and the future fetal
circulations
 Primitive Streak  arises from blastodisc in around Day
15
o Gastrulation is similar to chick and mouse, with
cells migrating through the streak to form the
mesoderm and a definitive mesoderm (which
replaces the hypoblast)
 Notochord fuses with endoderm  neurocentric canal
 Similar body plan formation with mouse, EXCEPT allantois 
evagination from hindgut and lined with endoderm
o Mouse: Allantois is composed of only the
mesoderm
MOUSE DEVELOPMENTAL GENETICS
Transgenic mice
 Transgenic- introduction of new genes into the germ line
 Standard method: Inject DNA directly into the pronucleus of
the fertilized egg
 Other methods:
-Knock-in
-use of integrase enzymes
 Screening for incorporation via Southern blot or polymerase
chain reaction (PCR)
 Correct temporal and regional expression so long as
sufficient flanking DNA sequences are included
 Bacterial artificial chromosomes (BACs) can accept more
flanking DNA than plasmids
 Transient transgenics- used to identify particular regulatory
sequences by embryo reimplantation
-Reporter genes: lacZ and Luciferase
 Express coding region of one gene under the control of
promoter of another enables ectopic expression
 Uniform expression
-Promotors for Housekeeping genes: cytoskeletal (β) actin
(Actb) or histone H4 (Hist4)
Embryonic stem cells
 Embryonic stem cells (ES cells)- cell lines showing
developmental pluripotency/ indefinite self-renewal
 Produced by putting blastocysts in tissue culture
 Suppress differentiation by growing ES cells on feeder
layers, in the presence of leukemia inhibitory factor (LIF) or
inhibitors for components of FGF and Wnt signaling
pathways
 Removal of differentiation suppressors will result to
embryoid bodies
 In vivo counterparts (ICM and early epiblasts) are NOT stem
cells
 Stem-cell property dependent on mutual upregulation of
transcription factors: OCT4, NANOG and SOX2 (pluripotency
factors) that repress expression of developmental control
genes
 Developmental control genes are maintained in a state of
“bivalent mark”
 When implanted into mice, ES cells form tumors called
teratomas
 When injected into mouse embryo blastocysts, it will result
to chimerism
Gene and enhancer traps
 Insertional mutagenesis- allows easy identification of
modified locus
 Gene traps= splice acceptor site + reporter gene +
selectable marker
 Reporter and selectable marker often combined into a single
gene: β-geo (fusion of E. coli lacZ and neomycin-resistance
gene)
 Rosa26- important gene trap mouse strain
 Enhancer trap is a gene trap that lacks a splice acceptor and
carries own minimal promotor
 Enhancer traps are usually not mutagenic. Main use is to
provide lines of mice needing particular tissues or cell types
highlighted by β-galactosidase
Gene Knockouts and Knock-Ins
Gene Knockout: One of the organism’s genes is made
inoperative; causes changes in a mouse’s phenotype
Gene Knock-In: Targeted insertion of a protein
coding cDNA sequence at a particular locus in an
organism's chromosome (*Transgenesis: Insertion site is not
defined)
Common Use: Creation of disease models
HOW TO CREATE A KNOCKOUT MOUSE:
1. Generate a targeting vector (Identifying which region
should be deleted)
- Targeting vector: a long stretch of DNA made up of
smaller pieces of DNA that have been joined together
- Positive selection kills all cells that were not
transformed, leaving a population of random and
targeted recombinants. Negative selection kills the
random recombinants, so that only targeted
(homologous) recombinants remain.
2. Insert the target sequence and select cells with the
insertion (Target vector is inserted into ES cells)
- ES cells: Embryonic Stem cells Barr body
- Targeting vector recombines with target gene and
knocks out one copy of target gene. (Result: cells with
knockout gene are neomycin-resistant and ganciclovir-
resistant, which means there’s no TK)
- Targeting vector recombines at a nonhomologous site
(Result: cells with random recombination are neomycin-
resistant and ganciclovir-sensitive, which means TK is
present)
3. Identifying ES cells with the correct gene knocked c) Teratocarcinoma
out (Only the ES cells that have had one copy of the target
gene knocked out remain)
- ES cells are heterozygous for the knockout mutation.
4. Injecting heterozygous knockout ES cells into a
developing embryo and transferring the embryo into
a mouse
- Heterozygous knockout ES cells become part of the
developing embryo
- Resultant mouse: chimeric mouse
OTHER TOPICS IN MOUSE DEVELOMENT
a) Nuclear transplantation and imprinting
1. Parthenogenetic embryo
-development from oocytes, without genetic contribution from
the sperm
-made by activation of the egg and suppression of second
polar body formation
-develop poorly because of inadequate formation of
exraembryonic tissues
2. Androgenetic embryo
-genetic material only from the sperm
-made by removing the female pronucleus and injecting a
second male pronucleus
-abundant extraembryonic tissue
-arrests at an early stage
b) X-inactivation
-process required to equalize the dosage of X-encoded genes
between female and male cells
-depends on the presence of X-inactivation center (Xic)
-contains gene Xist
- encodes nontranslated RNA
- works only in own chromosome
- its activity increases in inactivated chromosomes
-Tsix
-antisense gene
-overlaps Xist gene on DNA atrand
-active in maternally derived X chromosomes
-helps make maternal X resitant to early inactivation
- x-chromosome becomes hetrochromatic and inactive in
female mammals
-produces a mosaic
-some cells will express the gene in expression while
others will not
-seen in H253 strain of mice
-carry lacZ on the x chromosome
-malignant and transplantable tumors
-consists of transplantable tissues including undifferentiated
cells (embryonal carcinoma (EC) cells)
1. Spontaneous testicular teratocarcinoma
-arise in testes of fetal male mice
2. Spontaneous ovarian teratocarcinoma
-arise in female mice
-derived from oocytes that have completed the first meiotic
diviosion
3. Embryo derived carcinoma
-grafting early mouse embryos to extrauterine sites (kidney
or test

Imprinted genes
-genes expressed from either maternal or paterenal
chromosome

-E.g.
(IGF)-2 system
-prevention of parthenogenetic development
-active only in paternal chromosome
-silencing usually happens through the addition of methyl
groups during egg or sperm formation

IGF2
-growth factor promoting prenatal growth of embryo
-active only on paternal chromosome
-inhibitor active on maternal chromosome
Diseases involved in imprinted genes
Beckwith-Wildemann syndrome
-causes embryonic outgrowth and cancer

Wilm’s tumor
-loss of imprinting of IGF2
-pediatric cancer of the kidney