Limb Development Part 2

Question 1

Where is the ZPA activity located in the limb bud?

What happens if we graft the ZPA into the anterior region

Answer 1

Posterior in the limb bud

Posterior digits forming anteriorly

Question 2

How do the signals move from the ZPA?

Answer 2

Diffuse and decline

Question 3

What induces the formation of the ZPA

What is this molecule made from?

Answer 3

Shh

• Shh is induced by FGF8 from AER, restricted to posterior margin of the limb bud mesenchyme in hoxb8 region?
• Shh acts a morphogen to pattern the AP axis (short range?)
• Shh induces BMPs (2 & 7) – longer range morphogens to pattern tissues in the limb
• Shh induces FGF4 in AER, knocking this out disrupts digit formation
• Shh controls Hox phase 1 to 2 transition
• FGF4 and Shh mutual maintenance
• Wnt7a also required for maintenance

Question 4

Describe the Shh feedback loop involved in AP patterning of the limb bud

Answer 4

• On the LHS is an in situ hybridisation of Shh within the chick limb bud, it is restricted to the posterior mesenchyme of the limb bud.
• RA is needed for the expression of the transcription factor dHand which allows Shh to be expressed
• Shh acts through a BMP intermediate component Gre to permit the expression of FGF8 in the AER which is needed to maintain Shh expression
• Shh also singals through the transcription factor Gli3. Shh is inhiubitory of Gli3 from its activator form to Gli3 to its repressor form.
• The action of Shh leads to high levels of Gli3 in the posterior region of the limb bud and high levels of repressor in the anterior region of the limb bud.
• Wnt7a in the dorsal ectoderm is also required to maintain Shh expression

Question 5

Why do we have a series of complex feedback loops?

Answer 5

To coordinate limb size

Question 6

What feedback loops reduce Shh levels?

Answer 6

• BMP4 is expressed in the posterior limb territory and serves to inhibit FGFs
• BMP4 also turns on Grem1 with fast kinetics.
• Grem1 acts as an inhibitor of BMP4
• This inhibition removes the inhibtion of FGFs from BMP4
• FGF signalling now allows Shh which induces Grem1 expression
• As the limb grows the source of Shh moves further away so it can’t affect the induction of Grem1
• This Grem1 expression is then lost and the inhibition of BMP4 is also lost which can start to inhibit FGF then leads to the reduction of Shh signalling.

Question 7

BMPs are expressed in the interdigital regions, what happens if we remove the interdigital regions or use noggin as a inhibitor?

What do the digits form from?

What causes the breakdown of the interdigital mesoderm?

If we block apoptosis, what happens and how can we do this?

Answer 7

Inter-digital mesoderm specifies identity of digit anterior to it

If you remove the interdigital regions, you get anterior transformations of the digits (made of proliferative mesenchyme).

BMP induce apoptosis of the mesoderm between the digits (2,4, and 7). If block BMPs with a dominant negative receptor leads to syndactyly (joining of digits)

How do the digits stop themselves from being broken down? -> produce noggin a BMP inhibitor

Question 8

What are the anterior and posterior necrotic zones in the chick?

How is this different in the duck?

Answer 8

Digit formation by cell death
• Interdigital regions undergo cell death.
• The green regions in the chick are anterior and posterior necrotic zones which high levels of programmed cell death. Apoptosis occurs between the digits to form the foot
• In the duck limb, there is a smaller anterior necrotic zone and no posterior necrotic zone. The digital rays start to condense, there is limited apoptosis around the digits and between them which leaves the tissue that forms the web of the duct foot.
• BMP antagonists can form webbed feet as well.

Question 9

What are the 3 different modes of digit reduction in tetrapods.

Answer 9

1. Skink -> Shh levels decrease over time
2. Mouse -> BMP induced apoptosis
3. Mouse vs Cow -> differences in Ptch1 (receptor for Shh) due to differences in the regulatory module driving Ptch1

Question 10

What forms from the mesenchyme in the limb?

How is bone formed?

What stains the cartilage and what stains the bone?

Answer 10

• The cartilage is first laid down which is formed from the mesenchyme which will then be ossified by endochondral ossification
• These chick embryos have been stained with alcian blue to show the cartilage and alizarin red stain to show the bone tissue