Mesoderm formation in Xenopus Flashcards
The genes bra, myf5, gsc and vent1 are only expressed in which tissue? When? Do the overlap?
In the mesoderm at early gastrula stage. They do not overlap.
What is bra?
Brachyury: a t-box transcription factor expressed pan-mesodermally.
Where is bra expressed?
Always around the blastopore and always in the notochord.
Without bra the notochord would not form correctly. True or false?
True.
As cells involute over the blastopore they maintain bra expression. True or false?
False: involuting cells lose bra expression.
a) What is gsc?
b? Where is it expressed?
a) Goosecoid: a homeobox TSF
b) Above the dorsal blastopore lip on the future dorsal side
Do bra and gsc overlap in expression?
No.
What is myf5 responsible for?
Myogenic factor 5, TSF responsible for muscle formation.
The expression of bra, gsc, myf5 and vent1 becomes more refined as development progresses. True or false?
True.
In specification map experiments…
a) What happens if you isolate the animal hemisphere?
b) What happens if you isolate the vegetal hemisphere?
a) It only forms epidermis
b) Will not form endoderm, although endoderm genes are being transcribed. Cannot form endoderm without supportive mesoderm.
In specification map experiments…
a) What happens if you isolate the animal hemisphere?
b) What happens if you isolate the vegetal hemisphere?
a) It only forms epidermis
b) Will not form endoderm, although endoderm genes are being transcribed. Cannot form endoderm without supportive mesoderm.
In specification map experiments…
a) What happens if you isolate the dorsal equatorial region?
b) What happens if you isolate the ventral equatorial region? Will they differentiate?
a) Neural tissue forms
b) Bags of blood form full of RBCs/mesenchyme etc. They are supposed to form somites but WILL NOT differentiate in isolation
From the specification map experiments we can see 2 types of mesoderm are set up. What are they and which genes are expressed there? What is expressed in the equatorial region?
Dorsal mesoderm (neural tissue) expresses bra.
Ventral mesoderm (blood) expresses gsc
Equatorial regions express myf5 and vent1.
Outline the experiments by Pieter Nieuwkoop in 1960.
He combined the animal and vegetal hemispheres from different axolotl embryos
In the experiments of Pieter Nieuwkoop in 1960, what happened when he recombined the hemispheres at the start of gastrulation?
Nothing, he just got ectoderm mixed with yolk.
In the experiments of Pieter Nieuwkoop in 1960, what happened when he recombined the hemispheres at mid-blastula stage?
He got endoderm, mesoderm and ectoderm.
How did Pieter Nieuwkoop discover that mesoderm was derived from the animal cap?
He used a normal and an albino embryo.
What was Pieter Nieuwkoop’s conclusion from his experiments?
Mesoderm formed from the animal cap under induction from the vegetal cap.
Describe what changes were made when Nieuwkoop’s experiments were repeated in Xenopus.
The animal cap was labelled with fluorescein. All mesodermal tissues thus expressed fluorescein.
The vegetal cap makes absolutely no physical contribution to the mesoderm. True or false?
True: only involved in signalling.
Nieuwkoop’s experiments only work at which stage of development?
Blastula stage.
What happened in experiments whereby individual blastomeres from the vegetal hemisphere were combined with the animal cap?
Ventral blastomeres induced ventral mesoderm and dorsal blastomeres induced dorsal mesoderm.
How can the ventral/dorsal blastomeres be identified?
They have slightly different pigmentation due to cortical rotation.
D1 is a blastomere that can induce a secondary dorsal axis if transplanted into D4 position. D1 did not physically contribute to this secondary axis. What is D1 known as? Where is it found?
The Nieuwkoop centre: it is a specialised inductive centre for dorsal mesoderm. It is found on the extreme dorsal side.
Name 2 maternal TSFs.
VegT and beta-catenin.
Where is VegT found?
Throughout the vegetal hemisphere.
Where is beta-catenin found?
On the dorsal side of the embryo.
Where is the part of the embryo that vegT and beta-catenin overlap?
The Nieuwkoop centre.
Combining antisense oligonucleotides to prevent mRNA translation with grafting experiments showed that a) vegt and b) beta-catenin are essential for what?
a) Necessary for both dorsal and ventral mesoderm (vegT depleted embryo did not develop mesoderm)
b) Necessary for dorsal mesoderm induction (beta-cat depleted embryos did not develop dorsal mesoderm)
VegT and beta-cat are maternal TSFs. Can they initiate mesoderm formation directly?
No: they must activate zygotic TSFs
How was it proved that the molecules that induce mesoderm are diffusible proteins?
In grafting experiments a membrane was put between the animal and vegetal caps. Cells could not pass through it but signalling molecules could. Mesoderm still formed.
What does MIF stand for?
Mesoderm Inducing Factor.
What is activin?
A protein that induces notochord formation.
Why does activin cause the animal cap to change shape?
The animal cap forms ectoderm. The notochord is ectoderm. Activin causes it to elongate in convergent extension, thus changing the shape of the animal cap.
All mesoderm inducing factors produce various shape changes. True or false?
True.
What is TGFβ?
Transforming growth factor beta: a family of growth factor proteins.
What is activin’s homologue in mammals?
GDF3
What is FGF?
Fibroblast growth factor: a family of growth factor proteins.
All MIFs identified have been from either TGFβ or FGF. True or false?
True.
Is activin a MIF?
Yes.
Activin has differential effects depending on concentration. True or false?
True.
What effect does activin have in a) high and b) low concentration?
a) Dorsal mesoderm
b) Ventral mesoderm
If high activin induces dorsal mesoderm, what does super high activin induce?
Endoderm…
What is nodal?
Another MIF
What is bmp?
An MIF that only induces ventral mesoderm
All the TGFβ proteins appear to act in the same way. How?
High concentration produces dorsal mesoderm.
All the FGF proteins appear to act in the same way. How?
They induce ventral mesoderm EXCEPT in super high concentrations they will induce dorsal mesoderm.
All the TGFβ proteins appear to act in the same way. How?
High concentration produces dorsal mesoderm.
What kind of receptors do TGFβ have?
STK1 and 2: these bind to serine kinase 1 and 2
Why must STK1 and 2 be bound together by a TGFβ member?
STK1 is inactive without STK2.
What do bound STK1 and STK2 do?
Activate SMAD proteins the regulate gene transcription, e.g. bra.
Without STK1/2 signalling, what happens to embryos?
They are just balls of epidermis.
What is nodal?
A zygotic MIF
What was used to help deduce the role of nodal?
Cer-S: Cerberus short is a mutant version of Cerberus (a secreted binding protein). Cer-S targets nodal proteins and stops them binding their targets, thus can see what happens.
What happened in grafting experiments with Cer-S injected into the vegetal cap? What does this tell us?
The animal cap failed to produce mesoderm. Nodal is necessary for mesoderm induction.
Approx. how many nodal genes are there in xenopus?
6, there is only 1 in mammals.
How does a) vegT and b) vegT combined with beta-cat affect nodal transcription?
a) VegT alone causes low level transcription of nodal
b) Where vegT and beta-cat overlap in the Nieuwkoop centre, high level transcription of nodal is activated
Cells with beta-catenin are more sensitive to nodal. True or false?
True.
VegT is specific to anurans. True or false?
True.
What kind of receptors do FGFs have?
Receptor tyrosine kinase.
