Gene Expression (Nodal and Lefty = AVE/PS)- Gastrulation

Question 1

Nodal +/+

Answer 1

forms the mesoderm

Question 2

nodal is homologous to ____ in Xenopus

Answer 2

nodal

Question 3

How was nodal discovered?

Answer 3

it was discovered when retroviruses were used as mutagens to to infect Embryonic Stem cells.

1. retroviral RNA genome was reverse transcribed into DNA and integrated into the genome.
2. This replaced the exon sequence with that of the retrovirus.
3. ES cells carrying a large number of retroviral insertions were then introduced into the blastocyst.
4. Here the resulting mice would then carry mutations induced by the retrovirus.
5. Germ cells derived from ES cells propagate mutations into mice.
6. These heterozygous lines are then bred into F2 generations, where homozygotes are screened for interesting phenotypes.
7. When one such integration affected mesoderm development, cloning of retrovirus tagged locus identified nodal gene as the responsible gene.

Question 4

How is nodal expression amplified?

Answer 4

Through two positive feedbacks:

1. extra-embryonic tissues respond to low levels of nodal by expressing protease that cleaves the nodal precursor. Nodal protein is now mature and nodal protein function is amplified in the proximal epibast.
2. Nodal signaling activates nodal expression. There is a FoxH1 binding site in the first intron of the nodal gene. Normally FoxH1 is bound and recruits repressors, however, when Nodal signaling activates Smad2, the Smad2/4 complex displaces the repressors, and recruits activators leading to an activation of nodal expression.

Question 5

What can happen if the positive feedback is left unchecked?

Answer 5

Positive feedback is potentially dangerous, and if left unchecked, the entire embryo may become endomesoderm.

Question 6

Why does the positive feedback activate the negative feedback?

What is this negative feedback?

Answer 6

activates a negative feedback to moderate Nodal function.

lefty2, like nodal is a target of Nodal function. Lefty protein diffuses faster than Nodal, “overtakes” Nodal and limits its function. Lefty2 is Nodal’s antagonist.

Question 7

How can you test experimentally that lefty diffuses faster than nodal?

Answer 7

1. perform a reconstruction experiment
2. over express tagged nodal and lefty
3. follow protein diffusion

Question 8

What is the reverse genetic approach to determining gene function?

Answer 8

• create KO mice.

In contrast to forward screens, where random mutations are generated, and then screened for interesting phenotypes, in a knock out mouse, the experiment starts with a characterized and cloned gene. 


eg. lets look at Lefty2 that blocks nodal activity is also a target of nodal signaling. We want to know why is Lefty, an antagonist of Nodal signaling, also a target of Nodal signaling? To address this question, you would KO Lefty2 gene.

From the knockout phenotype, one can conclude that lefty2 is a negative feedback regulator of mesoderm induction because the phenotype showed that it prevents excessive mesoderm formation in the embryo [phenotype was an expanded streak].

Question 9

This gene is highly expressed in AVE

Answer 9

Lefty1

Question 10

This gene is highly expressed in PS and it’s expression depends on Nodal function.

Answer 10

Lefty2

Question 11

But how would you create a KO mouse?

Answer 11

Apply a reverse genetic approach:

1. all or part of the gene is deleted or replaced.
2. A knockout construct, carrying the mutation is made in vitro, and then introduced by electroporation into ES cells.

3. There is a requirement for
 1. a positive selectable marker- in this case neomycin resistance 
2. flanking regions of DNA homology on both sides of the selectable marker 
3. an optional (but useful) negative selectable marker (not discussed in lecture)

4. A way of determining whether the integration was homologous - i.e. a faithful replacement of the endogenous allele - Conduct a Southern blot/Polymerase chain reaction

5. Once the proper ES cell has been identified, it can be reintroduced into the mouse, and the chimera then bred to produce heterozygotes, and subsequently these are interbred to produce homozygotes.


Question 12

Nodal is an essential mesoderm inducer that acts through Smad 2/3 signaling - But how is it selectively deployed?


Answer 12

— Cerberus and Lefty1 block nodal activity in AVE and Lefty2 moderates nodal activity in the PS and proximal epiblast.

1. a small amount of Nodal signaling from the epiblast induces the expression of Cerberus and Lefty1 in the AVE….then they feed back to and prevent mesoderm induction locally.
2. Lefty2 is expressed in response to higher levels of nodal signaling in the proximal epiblast and primitive streak, and its action is to moderate the amount of mesoderm formed. 


These mechanisms illustrate both positive and negative feedback loops to establish the proper amount of mesoderm in the embryo.

Question 13

Phenotype of KO Cerberus and Lefty1

Answer 13

KO of cerberus and lefty1 === the mouse makes extra primitive streaks.


KO of cereberus = a little but of PS? !!!!!

Question 14

How does Nodal signaling become asymmetric?
 OR How does nodal signaling increase on one side of the cup, and lead to local formation of the primitive streak?

Answer 14

formation of the Anterior Visceral Endoderm, an extra-embryonic signaling center leads to expression of cerberus and lefty1 that are both nodal antagonists.

Question 15

What gene is expressed in the distal part of the egg cylinder OR DVE? AVE migration:

Answer 15

The distal part of the egg cylinder or Distal Visceral Endoderm, expresses the transcription factor Hex, Lefty and Cerberus.

Question 16

You marked the DVE cells with hex-GFP or Lefty-GFP or Cerberus - GFP reporter and want to see the active migration of these cells. What do you see?

Answer 16

DVE cells move away from the distal tip to the proximal lip of the egg cylinder cup

Question 17

Where in DVE are lefty and cerberus expressed? Why?

Answer 17

the earliest expression of lefty and cerberus is already biased to one side of the DVE, and this is thought to result from chance variation in nodal signaling in the ICM.

Increased nodal causes more cell proliferation in the VE, so local increase in nodal will “push” the VE away in the other direction. Once it has this bias from proliferation, it continues to move by migration.

Question 18

How does the AVE give rise to Anterior Nervous System?

Answer 18

As in other vertebrates, once the AVE underlies the epiblast, and expresses nodal, BMP and Wnt antagonists, that region is protected from nodal signals, BMP signals and Wnt signals, and can become the anterior nervous system.

Question 19

How is the AP axis established?

Answer 19

The AP axis is established by the asymmetry of the AVE, which underlies the future head, and determines the site of the beginning of mesoderm/ primitive streak formation, which in turn is the posterior part of the embryo.

Question 20

How is the DV axis established?

Answer 20

DV Axis is set up from the topology of epiblast and VE. As in Xenopus, an initially small asymmetry is magnified to establish a definitive asymmetry in gastrulation movements.

Question 21

Which organs exhibit a clear LR symmetry?

Answer 21

The embryo always turns in the same direction, and generates clear Left right asymmetry in a variety of internal organs: the heart, liver, lungs, gut, brain.

During formation of the node, the overlying primitive endoderm forms a gap, and the ciliated cells of the node are exposed.

The cilia point posterior, and rotate, inducing a leftward flow. This flow is thought to be causal in setting up the left side. This is another example of symmetry breaking. In this case, the source of the asymmetry is the molecular asymmetry of the proteins within the cilium, and this is overlaid on the anterior posterior polarity of the embryo (using the planar cell polarity pathway)- which sets up the cilia to point towards the posterior.

Question 22

Mutations that eliminate ciliary motility cause?

Answer 22

Mutations that eliminate ciliary motility cause randomization of the Left-right axis.

Question 23

The axis can be reversed by?

Answer 23

flowing fluid in the opposite direction