lecture 31 Flashcards
1
Q
Where does the sperm penetrate the egg?
A
- almost always in the animal cap
2
Q
Where does the dorsal part of the embryo form?
A
- area diametrically opposed to site of sperm entry
3
Q
What does presence of Beta-catenin in the nucleus indicate?
A
- indicative of pathway activation
- Wnt signalling
4
Q
What is the story so far?
A
- asymmetric distribution of maternal determinants leads to activation fo Wnt/Beta-catenin pathway and induction of dorsal genes (D-V axis)
- but this process is inter-linked with mesoderm induction and specification of anterior-posterior (A-P) axis
5
Q
What are mesoderm inducers?
A
maternal determinants in vegetal cells
- Vg1 (TGF-beta family of growth factors) (remains in vegetal pole)
- Veg-T (remains in vegetal pole)
- these act on animal cap cells - induce activation of other TGF-beta genes
- some shifted by cortical rotation to be in the dorsal part of the embryo
Earliest zygotic genes expressed in vegetal cells:
- Xnr5, Xnr6, Derrière (nodal-related proteins; TGF-beta family)
- expressed in animal cap cells due to induction from vegetal cells
Experiments
- deplete Veg-T –> failure of Xnr expression and mesoderm formation
- rescue mesoderm formation by injecting mRNA for Xnr proteins
- know that two signalling activated in early blastula embryo
- Wnt, nodal-related
- wnt reinforces the production of nodal –> get very high levels in the more dorsal vegetal cells
- this gradient of nodal specifies the mesodermal structures induced
6
Q
Why are xenopus/zebra fish such good models for experimental embryology?
A
- lay lots of eggs
- relatively large embryos accessible in terms of injection procedures
- injection of mRNA
Experiment:
- inject mRNA for growth factors or mutant receptors in early Xenopus
- inject antisense RNA to knock-down gene expression
- assay for mesoderm formation or axis duplication
- inject mRNA encoding growth factor or receptor into both cells of 2 cell embryo
- can inject or KO canditate genes
- can wait for hours or a day to get results as opposed to months
7
Q
Do TGF-beta signals induce mesoderm?
A
Experiment:
- inject mRNA for TGF-beta superfamily in early xenopus embryos
- nodal/activin signals –> induce dorsal mesoderm
- i.e. SUFFICIENT
- BMP signals –> ventralise mesoderm but only after mesoderm is induced (weren;t sufficient to induce mesoderm)
- these two pathways activate different intracellular mediators
- Nodal TGF-beta activin: smad 2, smad 3
BMPs activated Smad1, Smad5, Smad8 - actual molecule that activates nodal/activin pathway are the nodal-related proteins
Experiment:
- inject mRNA for mutant dominant-negative activation/nodal receptors in early Xenopus embryos –> no mesoderm
- i.e. REQUIRED
- but other factors also required (e.g. FGF)
- no signal from mutant receptor even when dimerised with wild-type receptor subunit
- need FGF to have competence to respond to TGF-beta
8
Q
What multiple signals specify endoderm, mesoderm and ectoderm?
A
- VegT (maternal) required for endoderm specification of vegetal cells
- VegT induces expression of Nodal proteins (zygotic) that induce formation of mesoderm
- Animal cap cells express:
- ectodermin (maternal), gradient inhibits mesoderm induction; a ubiquitin ligase - targets Smad4
- Foxl1e (zygotic) specifies ectoderm – epidermis and stops ‘mixing’, also regulates a different cadherin expression from mesoderm, cells with similar cadherins tend to stick together
- ectoderm has a repressive effect on mesoderm induction
- ectodermin antagonises TGF-beta signalling
- smad4 is required to bind to 2 and 3 - without it doesn’t matter how much TGF-Beta signalling you have no gene transcription will occur
9
Q
What is the current gradient model?
A
- interaction of Wnt signals with Vg1/VegT results in a gradient of Nodal signals from dorsal–ventral
- highest nodal occurs in Nieuwkoop centre –> Dorsal mesoderm (organiser)
Beta catenin + VegT/Vg1 –> nodal-related HIGH –> dorsal mesoderm and ‘Organiser’
VegT, Vg-1 –> Nodal-related LOW –> ventral mesoderm
10
Q
What is the Nieuwkoop centre?
A
- graft of dorsal blastomeres (or Beta-catenin mRNA injection) can induce ventral to become dorsal and induce a complete new body axis including nervous system (neural tube and brain)
- took dorsal blastomere and transplanted it to ventral side of the embryo
- resulted in mirror image - double axis
- lead to the concept of ‘organiser’ and what specifies head-to-tail axis
- get same result if you inject Beta-catenin into ventral blastomere
11
Q
What did Hans Spemann/Mangold discover?
A
- Spemann won the Nobel prize in 1935 for this work
- Mangold’s PhD thesis but she died in a home accident prior to award and prior to thesis publication
- a piece of the upper blastopore lip of an amphibian embryo undergoing gastrulation exerts an organising effect on its environment in such a way that, if transplanted to an indifferent region of another embryo, it causes there the formation of a secondary embryonic anlage (precursor)
- such a piece can therefore be designated as an Organiser
12
Q
What is the spemann organiser?
A
Induction
- graft of dorsal blastopore lip has the capacity to induce a complete new body axis including nervous system (neural tube and brain)
- what is the role of the Organiser in A-P axis formation?
13
Q
What is the role of the organiser in gastrulation?
A
- induced by high Nodal from Nieuwkoop centre
- differentiaties into dorsal mesoderm (e.g. notochord)
- dorsalises surrounding mesoderm to form paraxial mesoderm (somites)
- dorsalises ectoderm –> nerual tube formation (neurulation)
- initiates gastrulation movements (A-P axis)
- mesodermal cells converge and move towards dorsal blastopore lip (also ventral, but most dorsal)
- then move anteriorly towards head region
14
Q
How does gastrulation occur in xenopus?
A
- complex series of morphogenetic movements
- involution – “rolling in” of endoderm/mesoderm
- convergent extension of mesoderm
- epiboly of ectoderm
- bottle cells form at site of blastopore (vegetal cells)
- become very involuted and very large, elongated cells
- have a constricted cytoplasm likely due to constriction of actin filaments
- considered to be initiators of gastrulation
- involute and form blastopore lip
- rolling and movement of cells towards blastopore lip
- progress underneath in this blastocoel space and migrate anteriorly toward head region of the embryo
- bottle cells become detached and lead mesoderm to migrate under the embryo
- at the same time you have epiboly: animal cap cells are proliferating and moving around the whole embryo
- pulling on a beanie = epiboly
- poking finger into balloon without poking it = convergent extension
- as this progresses you get a rod of mesodermal cells lining up along what will be body axis
- eventually comes to anterior part of embryo
- epiboly leads animal cap cells to cover almost all the embryo
- only thing left is yolk plug (eventually disappears - forms anus?)
- archenteron forms during involution - forms gut
endoderm, mesoderm and ectoderm now in the places they need to be to form all structures of the embryo
head end and tail end specified
15
Q
What is involution?
A
- first stage is apical constriction of cells (bottle cells)
- involution of subsurface marginal cells (migratory cells) leads to pulling in of the superficial cells (bottle cells)
- cells migrate along the roof of the blastocoel and form the archenteron
- bottle cells contribute to the roof of the archenteron
- as the endomesoderm migrates it undergoes convergent extension
