germ layer formation and gastrulation (L8)

Question 1

What organism was mainly used to study germ layer formation?

Answer 1

Mainly understand germ layer formation and gastrulation using Xenopus. This is because Xenopus were readily available when the research was being done (because they were being used as pregnancy tests in GPs - oestrogen from wee makes them lay eggs)
However, the same molecules are thought to operate in chicks/humans - the genes are conserved.

Question 2

How do you transform an apparently uniform group of cells into cells with a defined identity and arranged in axes?

Answer 2

1. Set aside ‘top cells’ Vs ‘bottom’ by differentiation - at the 4 cell stage, the embryo divides across the equator to make 4 top cells and 4 bottom cells.
2. The bottom cell signals to the top cells so they start differentiating further.
3. The 4 top cells become the animal hemisphere (epiblast in humans and chicks) and eventually form the 3 germ layers
4. The bottom 4 cells become the vegetal pole (hypoblast in humans and chicks)

Question 3

How is the oocyte polarised?

Answer 3

Particular components are specifically found in the vegetal hemisphere e.g. veg-T. The egg is polarised in the Xenopus because of gravity, and because of interactions with the placenta in mammals. Different cytoplasmic determinants have sunk to one part of the egg. This leads to cells that come from this region is different and has specific factors localised/activated.

Question 4

What happens to the Xenopus oocyte post-fertilisation

Answer 4

the Xenopus divides to a compact ball of cells (morula). Early division planes separate the vegetal component expressing cells from the non-vegetal expressing animal hemisphere cells. All the progeny of these different groups will be different - the TFs act autonomously.

Question 5

How are the germ layers specified?

Answer 5

A TF (VegT) localised to the nucleus in the vegetal cells binds to the promoter of, and activates transcription of the gene Nodal. This codes for a secreted morphogen which can diffuse out of vegetal cells and into the animal cells. The animal hemisphere cells that contain receptors for nodal, the nodal signal transduction pathway is activated. Cells close to nodal will change their fate - instead of becoming ectoderm, they will become either mesoderm or endoderm (depending on how close they are) The ones closest become endoderm and the ones farther away become mesoderm. Shows how important the veg pole is - if there was no nodal, all cells would become ectoderm and it wouldn’t be good :// Cells only respond if the pathway for the morphogen can be induced.

Question 6

Explain what role Wnt signalling has in gastrulation (look at diagram in notes if you don’t get it xx)

Answer 6

The Wnt signalling pathway is activated on one side of the embryo - classically we call this the dorsal side, but at this point in time it marks the site where gastrulation movements will begin. So this region marks the future dorsal part of the body axis. The sperm always fertilises the animal side of the egg, opposite the sperm entry point, the future organiser will form. When the sperm enters the egg, which consequently initiates rotation of the cortex. This activates Wnt signalling pathway on this side of the embryo (the future dorsal side). So the dorsalising factors are shifted to 1 side and the oocyte no longer has any symmetry.

Question 7

What is Nieuwkoop centre?

Answer 7

In the dorsal cells, there is an overlap of Nodal and nuclear beta-catenin, this region is called the Nieuwkoop centre. It has important properties. Beta-catenin is stabilised and transported into the nucleus. IN the Nieuwkoop centre, you get higher nodal activity. Nodal acts like a morphogen so can induce different fates and different concentrations. High nodal activity from the N centre is what induces the organiser. lower nodal leads to the induction of ventral mesoderm.

Question 8

Why does combinations of high nidal and active Wnt (beta-catenin) induce the organiser?

Answer 8

Genes like goosecoid (Gsc) are only transcriptionally active via both a nodal and downstream effector (Smad2/4) and a Wnt/beta-catenin effector. Critically, the induction of the organiser means that these cells start to express specific transcription factors like siamois, goosecoid and Xnot, Xlim1. Different types of mesoderm are induced that express specific transcription factors. Brachyury is induced in response to low levels of nodal (a bit in mesoderm and ectoderm)

Question 9

How do TFs expressed in the organiser cells alter surrounding cells?

Answer 9

The TFs expressed in the nucleus of the cells in the organiser/node e.g. Gsc and siamois now begin to bind to promoters (in an intrinsic manner) to alter the behaviour of the cells in which they are expressed - including changes to migration and differentiation.
Organiser/node cells begin to differentiate into axial mesoderm. These axial mesoderm cells migrate collectively in a process called convergent extension. Siamois/Gsc is expressed at fractionally differently levels in different cells in the organiser, so different types of axial mesoderm cells form, including prechordal mesoderm and notochord. These events are going to drive A/P axis formation.

Question 10

What is gastrulation driven by?

Answer 10

Gastrulation will be driven by the movement of cells, a movement that begins at one end, and so defines the posterior, and moves anteriorly.