lecture 31

Question 1

Where does the sperm penetrate the egg?

Answer 1

• almost always in the animal cap

Question 2

Where does the dorsal part of the embryo form?

Answer 2

• area diametrically opposed to site of sperm entry

Question 3

What does presence of Beta-catenin in the nucleus indicate?

Answer 3

• indicative of pathway activation

- Wnt signalling

Question 4

What is the story so far?

Answer 4

• 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

Question 5

What are mesoderm inducers?

Answer 5

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

Question 6

Why are xenopus/zebra fish such good models for experimental embryology?

Answer 6

• 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

Question 7

Do TGF-beta signals induce mesoderm?

Answer 7

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

Question 8

What multiple signals specify endoderm, mesoderm and ectoderm?

Answer 8

• 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

Question 9

What is the current gradient model?

Answer 9

• 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

Question 10

What is the Nieuwkoop centre?

Answer 10

• 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

Question 11

What did Hans Spemann/Mangold discover?

Answer 11

• 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

Question 12

What is the spemann organiser?

Answer 12

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?

Question 13

What is the role of the organiser in gastrulation?

Answer 13

• 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

Question 14

How does gastrulation occur in xenopus?

Answer 14

• 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

Question 15

What is involution?

Answer 15

• 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

Question 16

What does convergent extension involve?

Answer 16

• mediolateral intercalation – directional (narrows along one axis, extends along other)
• ring of cells that are converging at dorsal end
• these cells need to be able to intercalate with each other
• one of these processes involves medio-lateral intercalation

Question 17

What is gastrulation in the chick?

Answer 17

• very different morphology but same processes of ingression, convergent extension and epiboly
• converge onto primitive streak and migrate

Question 18

What is epiboly?

Answer 18

involves:

• radial intercalation – multilayered epithelium into single layer –> increased surface area
• cell proliferation