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INTRODUCTION
Frog ovary:
- Consists of oocytes surrounded by layers of follicle cells and blood vessels
- Oogonia continue to divide and become Primary Oocytes after their last mitotic division
*Growth of oocyte takes several months
- Oocyte nucleus
-> AKA Germinal vesicle
-> very large
- Chromosomes
-> AKA Lamp-brush chromosomes
-> four-stranded bivalents characteristic of meiotic prophase
-> remain transcriptionally active during oocyte growth
-> display numerous protruding loops of chromatin
Previtellogenic (preyolky) oocyte begins as transparent and becomes opaque due to yolk granules
*When the oocyte is fully grown, transcription ceases but protein synthesis and degradation
continues.
Animal-vegetal polarity arises
-> Mitochondrial cloud:
- AKA Balbiani Body
- Precursor of germ plasm: its location determines the future vegetal pole of the egg.
MATURATION PROCESS
Achieved in response to gonadotrophins
FERTILIZATION:ZYGOTE
Male clasps female and fertilizes egg as they emerge from the cloaca.
Secondary oocyte becomes fertilized eggs (zygote)
Rise in intracellular calcium
1. brings destruction of cytostatic factor -> breakdown of cyclin -> progression into second
meiotic division -> release of second polar body
2. Cause exocytosis of cortical granules near egg surface -> allows egg to rotate freely under
gravitation and bring animal hemisphere uppermost.
NORMAL DEVELOPMENT
Normal Development
Developmental substages:
Cleavage
Gastrulation
Neurulation
*Complete development of Xenopus: 24 hours at 24 degrees Celsius
> Nieuwkoop and Faber: devised a numerical stage series
Stage 8: Mid-blastula
Stage 10: Late gastrula
Stage 13: Early Neurula
Stage 24: End of neurulation
Cortical Rotation: cytoplasmic rearrangement initiated by sperm entry
- rotation of the egg cortex relative to the interior
- associated with the transient appearance of an oriented array of microtubules in the vegetal
hemispheres
- leads to the REDUCTION IN THE PIGMENTATION OF THE ANIMAL HEMISPHERE
(dorsal side, opposite to the sperm entry point)
Dorsal Determinant: moved from the vegetal pole to the dorsal side
- ensures that the dorsal structures will develop opposite the point of sperm entry
Grey Crescent: similar pigmentation in other amphibian species; surface feature
A. CLEAVAGE
First Cleavage: vertical; separates left and right hemispheres
Second Cleavage: vertical (right angle to the first cleavage); separating dorsal from ventral halves
Third Cleavage: equatorial; separating animal to vegetal hemispheres
Blastomeres: large cells resulting from early cleavage divisions
Blastocoel: cavity forms in the center of the animal hemisphere
Blastula: resulting embryo form cleavage division
>Outer surface of the blastula: consists of original oocyte plasma membrane
>Complete network of tight junctions: around the exterior cell margins that seals the blastocoel from the
exterior and renders the penetration of almost all substances highly inefficient.
>Oocyte: permeable up to stage of maturation to substances (amino acids and sugars)
>Internal cells of blastula: connected by cadherins and are readily dissociated by removal of calcium ions
from the medium
>Desmosomes: are not found until neurula stages
Gap Junctions: connects all the cells of early cleavage stages
Mid-Blastula Transition/MBT: cleavage continues rapidly for 12 hours
- actually occurs at the late blastula stage
- RATE OF CLEAVAGE SLOWS DOWN, SYNCHRONY OF CELL DIVISION IS LOST, and
INCREASE IN THE STRENGTH OF INTERCELLULAR ADHESION
- significant transcription of zygotic genome commences: possible detection of low level
transcription of some genes during the cleavage stage.
> T-box gene brachyury: visualization of entire mesoderm which is needed to upregulate later
mesodermal genes and to control gastrulation movements.
Organizer Region/Spemanns Organizer: in the dorsal sector that is characterized by the expression of
a variety of transcription factor genes (siamois, goosecoid, not & lim1)
siamois: involved in the original formation of the organizer
goosecoid: needed for gastrulation movements
vent1 and vent2: homeobox genes that expresses by ventral mesoderm that occurs in nested
pattern
vent1+2: specify lateral plate
vent2: specify somatic regions
mix1 and sox17: visualization of endoderm that are later required for upregulation of various
genes of particular endoderm derived tissues.
B. GASTRULATION
- phase of morphogenetic movements
Marginal Zone: belt of tissue that will become internalized through the blastopore.
>Start of Gastrulation: marked by the appearance of a pigmented depression in the dorsal vegetal
quadrant.
>Blastopore: becomes elongated laterally and soon becomes a complete circle
>Dorsal Lip: dorsal segment
>Ventral Lip: ventral part
>Bottle cells: elongated cells that are present around the blastopore
Several components of gastrulation movements
1. Epiboly: active expansion of animal hemisphere that eventually covers the whole embryo surface
2. Invagination: movement of the marginal zone. Starts on the dorsal -> spreads to the lateral and
ventral side -> blastopore is circular
Archenteron: cavity formed by invagination and expands at the expense of the blastocoel
3. Involution: mesoderm separates from endoderm and ectoderm
4. Convergent Extension: elongation of dorsal axial mesoderm that is an active process of
intercalation which helps drive the internalization of the marginal zone and the closure of the
blastocoel
5. Movement of the ventrolaterla mesoderm: towards the dorsal midline
>Rho and rac: small GTP exchange proteins that is required in convergent extension
- dominant (-) versions: block the process
- activated by Wnt planar polarity pathway with wnt5a: dominant ligand
>Paraxial protocadherin: cadherin-type adhesion molecule that is expressed in the paraxial mesoderm and
helps to generate activation of rho and rac through its cytoplasmic domain.
>Wnt Ca pathway: regulates cell adhesion during gastrulation, with the dorsal cells undergoing
convergent extension -> INCREASE INTRACELLULAR CALCIUM
Anteriorposterior axis: runs in the leading edge of mesoderm (anterior) to the residual blastopore
(posterior).
Exogastrulation: endomesoderm evaginates from ectoderm; dumbbell-like structure
- Ectoderm and endomesoderm: normally patterned
- Central Nervous System: substantially defective
- NO TAIL
D. FATE MAPS
- published for amphibian embryos at stages form the fertilized egg to the end of gastrulation
Vital staining: neutral red or Nile blue which applied to embryo surface from a small fragment of
impregnated agar
Lineage labels: injection of horseradish peroxidase or fluorescein-dextran-amine which can fill the whole
cells and not diffuse
DiI: for surface marking that do not become diluted and so remain clearly visible for several days
Expression pattern
In situ hybridization
Biological activity
Effect of specific inhibition in vivo
Injection of materials
Microsurgical isolation of explants
Ultraviolet irradiation
Gain of Function
In situ hybridization
For expression studies
Detection of a specific RNA, or occasionally DNA, in a biological specimen by
hybridizing to a specific probe with a suitable detection method
Probe: a labeled and antisense nucleic acid that can be used to detect the
complementary nucleic acid by hybridization
The same plasmid can be used for preparation of the in situ probes required by
transcription from te oppositely oriented promoter
Transgenesis
Late development
Injected RNA may have been degraded
Plasmid DNA is incorporated into swollen sperm heads
Injected into unfertilized eggs
DNA is injected into fertilized eggs in the presence of a transposase or a rare-
cutting restriction endonuclease
A GFP coding sequence is incorporated into the transgene
To identify the transgenic embryos by their green fluorescence
Each individual transgenic embryo will have a different insertion site and copy
number
Most founders are used as founders
Causes some variability of biological behavior compared with a pure-
bred transgenic line
Effects of overexpression of a specific RNA are often not very informative due to the
nonspecific nature of the defects observed
2. Autoinduction
Embryo is injected with a component of the mesoderm- or neural-inducing systems
Animal cap is explanted
Animal cap: animal pole region of the blastula
Explanted animal cap will autonomously undergo induction
Some or all of the cells in the cap make and secrete the factor
All the cells are competent to respond to it
Untreated animal caps
spherical balls of epidermis
Induced to form axial tissues
undergo convergent extension
become very elongated
Induced to form ventral-type tissues
swell
form translucent vesicles
Observed under the dissecting microscope
Simple and convenient
4. Ultraviolet rescue
It is possible to create embryos with no axial structures by UV radiation
Injection of mRNA can restore a supposedly lacking part of an embryo which was treated
with UV radiation
Formation provides a semiquantitative measure of the degree of rescue
Easily scored by visual inspection
Loss of Function
Determinants
1. Ventral determinant
Established at oogenesis due to mRNA localization at vegetal cortex
Formation of endoderm
the source of mesoderm-inducing signals forming the mesoderm
2. Dorsal determinant
formation of the organizer at the region of which becomes the dorsal lip
Initially at the vegetal pole but shift towards the dorsal pole due to cortical rotation
Organizer become source of signals (neural plate and pattern the mesoderm)
3. Germ plasm
visible in electron microscope
patch of fibrillar granular material
Contains cat2 mRNA
express primordial germ cell marker
Appear: mitochondrial cloud (balbani body) of stage II oocyte
End up in: Vegetal cortical region of mature oocytes and early embryo
Evidence of Determinants
1. Twin (Lateral)
Separation of two blastomeres at 2-cell stage
Suggest both halves have determinants
3. Rotation Movement
Due to relative displacement of surface-tethered microtubules relative to internal dynein
Cortical Rotation
Dorsal determinant is moved from vegetal pole to dorsal pole
Depends on the array of parallel microtubules, rotation and associated motor protein,
kinesin and dynein
Dorsal Determinant
Consist of component of the conanical Wnt signal transduction pathway
Treatments:
1. Hyperdorsal embryos
produced by treating early blastulae with lithium salts
resemble radially symmetrical heads
2. UV-ventralized embryos
Arise when mesoderm causes a development as organizer tissue
Rescued to normal pattern by localized injection of lithium
3. Ventral injection
Injection of lithium in the ventral side of a normal embryo will induce a secondary dorsal
axis
4. Mechanism
A. Lithium
Inhibitor of gsk3
Mimics the effects of Wnt signaling
Additional effects through inhibition of the inositol phosphate pathway
B. -catenin
Immediately target Tcf transcription factors
C. Tcf
Converted from an activator to a repressor by association with -catenin
Tcf3 has its repressive activity neutralize
D. Net result
Upregulation in the dorsal sector organizer genes
1. Encoding many transcription factors
A. Goosecoid
2. Many signaling molecules
A. (BMP) Bone morphogenetic protein inhibitors
PROPORTION REGULATION
o If a material is removed from the embryo, the pattern that eventually forms does not have a
gap but rather consists of a reduced-scale version of the normal pattern.
o Pattern is usually not perfect but certainly can tend toward the normal
o Removal of molecular components that regulate proportion such as BMP 2,4,7 and ADMP
leads to total neutralization
ADMP- antidorsalizing morphogenetic protein; a bone morphogenetic (BMP) type inducing factor
ANTEROPOSTERIOR PATTERNING
o Neural-inducing activity is shown both by the organizer and by the axial mesoderm into which the
organizer develops.
o Posterior organizer or posterior axial mesoderm induces both brain and spinal cord
o Posterior signal controls anteroposterior patterning during the formation of the central nervous
system (CNS)
o The two domains within the organizer in the initial anteroposterior pattern are derived from the
different ratio of -catenin arising from cortical rotation and nodal-related signaling arising from
mesoderm induction
*goosecoid will induce expression of anterior-type genes from ectoderm, such as otx2 (fore/ midbrain)
and ag1 (cement gland)
Posterior part- will later become the notochord and somites and, during gastrulation, it elongates
considerably by convergent extension.
*homeobox gene not and the T-box gene brachyury will induce expression of both anterior- and
posterior-type genes from the ectoderm (e.g. both otx2 and Hox genes), and its posteriorizing activity due
to secretion of FGFs and Wnts. Together these upregulate a group of homeodomain transcription factors
encoded by the cdx genes and these in turn upregulate the posterior Hox genes of paralog groups 613.
Evidence that FGF and Wnt signaling is required to induce the trunktail region:
1. If animal caps are treated with the BMP inhibitor noggin, then only anterior-type neural genes are
induced, but addition of Wnt or FGF will also induce posterior neural genes
2. Overexpression in embryos of a dominant negative FGF receptor that inhibits endogenous FGF
signaling, or of the dick- kopf Wnt inhibitor, will prevent formation of the posterior.
3. Overexpression or inhibition of retinoic acid does have effects on the pattern but in Xenopus largely
confined to the hindbrain
FGF- effects mostly felt on the trunk-tail region
Wnt- effects reach on the hindbrain region; controls engrailed [midbrain-hindbrain region]
ORGANIZER GRAFT
o All three of the processes, dorsalization, neural induction, and anteroposterior patterning are
shown here
o A piece of tissue from above the dorsal blastopore lip is implanted into the ventral marginal zone
leading to the formation of a double dorsal embryo.
o The notochord of the ectoderm above the graft becomes induced to form a second neural tube. As
gastrulation proceeds, both host and graft axes form progressively more posterior parts.
o The secondary axes arising from organizer grafts often lack a head, but if the graft includes the
deep anterior region of the organizer then this will emit cerberus and dickkopf and induce a head in
the overlying ectoderm.