XENOPUS
XENOPUS
XENOPUS
● 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.
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.
● 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 regio 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 elongted
● Induced to form ventral-type tissues
→ swell
→ form transluscent 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
A. Cause stabilization of β-catenin
➔ Injection of Wnt mRNA
1. Rescue formation of a dorsal axis in UV-ventralized embryo
2. Induce a second dorsal axis in normal embryo
➔ Β-catenin
1. Can be seen by immunostaining to enter nuclei in dorsal side
2. Essential role is obtained by antisense oligonucleotide mediated ablation of β-catenin mRNA
from oocyte
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. Β-catenin
● Immediately target Tcf transcription factors
C. Tcf
● Converted from an activator to a repressor by association with β-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
VI. INDUCTIVE INTERACTIONS
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 β-catenin arising from cortical rotation and nodal-related signaling arising from
mesoderm induction
Anterior part- will become anterior ectoderm and prechordal mesoderm
*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 a nd the T-box gene brachyury will induce expression of both anterior- and
posterior-type genes from the ectoderm (e.g. both otx2 a nd 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 6–13.
Evidence that FGF and Wnt signaling is required to induce the trunk–tail 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.