Lecture 8 Germ layer induction and gastrulation

Question 1

What do morphogens induce and how?

Answer 1

• Morphogens induce different transcriptional profiles, in a concentration dependent way

Question 2

How is cell fate determined

Answer 2

Through extrinsic and intrinsic factors

Question 3

How does development occur

Answer 3

Through a combination of cell proliferation, differentiation, migration and tissue morphogenesis

Question 4

Define morula

Answer 4

A uniform group of cells

Question 5

When do most miscarriages occur?

Answer 5

In the 1st 12 weeks due to failed germ layer formation/gastrulation

Question 6

What is the first step in germ layer formation/gastrulation

Answer 6

Set aside ‘top’ cells versus ‘bottom’ cells by differentiation

Question 7

How does the first step in gastrulation occur x3

Answer 7

• Different cytoplasmic determinants i.e. heavy components in the cytoplasm of the original cell ‘sink’ to the bottom under the effect of gravity (in mammals, one side of the embryo implants into the uterine wall to define the direction i.e. due to interaction with the placenta).
• Initiate early differences in future bottom (vegetal hemisphere) versus top (animal hemisphere) cells
• Oocyte (egg) is already polarised, even before fertilisation. Particular cell components are specifically found in the ‘vegetal hemisphere’

Question 8

Do the top or bottom cells differentiate into the 3 germ layers and why?

Answer 8

The top cells

The bottom cells signal to the top cells to differentiate further forming 3 germ layers

Question 9

What occurs in the 3rd division of the morula

What is the important thing here

Answer 9

Horizontal cleavage
4 cells on top and 4 on bottom
Gives animal and vegetal cells
Important thing is breaking symmetry

Question 10

What is the second step in germ layer formation

Answer 10

A blastocoel (hole) forms in the animal hemisphere due to changes in cell adhesion and polarity. This is known as a blastula

Question 11

Which hemisphere do the germ layers come from

Answer 11

The germ layers all come form the animal hemisphere = non Vg1-expressing part

Question 12

How do the 3 germ layers arise from the animal hemisphere?

Answer 12

Mesoderm induction
VgT TF family is localised to V hemisphere cell nuclei and binds to the promotor of and activates transcription of the gene Nodal

Question 13

How do we know VgT is expressed vegetally

Answer 13

In situ hybridisation - make antisense probes to see if particular gene is transcribed in a particular cell to give a white dot

Question 14

What does VgT code for

Answer 14

A secreted morphogen i.e. Nodal

Question 15

What occurs when Nodal is translated

Answer 15

It diffuses out of vegetal cells into animal cells which contain R for it to activate the Nodal signal transduction pathway

Question 16

What is the result of activation of nodal

Answer 16

cause that cell to change its behaviour (cell proliferation/migration/differentiation).

Question 17

In cells seeing high levels of Nodal, what do they differentiate into

Answer 17

Endoderm

Question 18

In cells seeing mid levels of Nodal, what do they differentiate into

Answer 18

Mesoderm

Question 19

In cells where the Nodal signalling pathway is not activated what do cells differentiate into

Answer 19

Ectoderm

Question 20

Why is the vegetal hemisphere so important

Answer 20

It signals to animal hemisphere to induce mesoderm and endoderm, otherwise animal hemisphere would just form ectoderm

Explains how 3 germ layers form

Question 21

What does VgT signalling not explain

Answer 21

How a particular part of the embryo arises that will drive gastrulation and axis formation

Question 22

Explain DV axis formation

Answer 22

1. Dorsalising factors sink to the vegetal pole
2. Sperm fertilisation only on animal side
3. Causes a 30 degrees cortical rotation
4. Dorsalising factors shift to a new position
5. Dorsalising factors activate Wnt sig pathway
6. This marks future dorsal/posterior part of the body and is the site where gastrulation is intiated
7. Beta-catenin enter the nucleus to act as a TF
8. In the Nieuwkoop centre, VgT and beta-catenin overlap
9. Co-expression of β-catenin and VgT turns on Nodal at very high levels. The mesoderm and endoderm, induced in response to Nodal signalling, at this point is slightly different: it’s Spemann’s organiser (cells receiving Wnt and very high Nodal).

Question 23

What is the organiser called in humans/chicks

Answer 23

Node or Hensen’s node

Question 24

Why does this combination of high Nodal and active Wnt induce the organiser? ie cells with a particular transcriptional signature?

Answer 24

A proximal element to the goosecoid (Gsc) gene is bound by β-catenin and Wnt; a distal element is bound by TGF-β/Nodal. Genes like goosecoid (Gsc) are only transcriptionally activated via BOTH a Nodal downstream effector (Smad2/4) and a Wnt/beta catenin downstream effector.

Question 25

Where are Vg-1 expressing cells found

Answer 25

Below the blastocoel

Question 26

Vg-1 negative cells are found where

Answer 26

On the side of the blastocoel

Question 27

What does the embryo proper develop from

Answer 27

Vg-1 negative cells

Question 28

When does the morula separate into top epiblast and bottom hypoblast layers

Answer 28

12 days