Lecture 15 Vertebrate limb development

Question 1

Thumb is…

Answer 1

Anterior

Question 2

Little finger is…

Answer 2

Posterior

Question 3

Back of hand is…

Answer 3

Dorsal

Question 4

Palm of hand is…

Answer 4

Ventral

Question 5

How many limbbuds are there in the chick?

Answer 5

4 because they develop with bilateral symmetry

Question 6

What do the limbbuds develop next to?

Answer 6

Somites

Question 7

Essentially, how does the AER form?

Answer 7

The limb buds grow out into the surrounding skin, thickening the skin to form the AER which is important for future patterning of the limb bud.

Question 8

How many TF specify limb identify? What is their collective name?

Answer 8

2 T-box TF

Question 9

Describe the different T-box TF

Answer 9

• Tbx5 is only expressed in the forelimb buds to specify wing identity
• Pitx1 and Tbx4 are only expressed in the hindlimb buds to specify leg identity
• Tbx5 represses Tbx4 which is why Tbx4 isn’t expressed in the forelimb bud
• Pitx1, which is restricted to the P limb bud, induces expression of Tbx4

Question 10

How does the Hox code and RA gradient specify limb identity

Answer 10

• Early in embryogenesis there is a gradient of RA set up from P  A
• This sets up the netted expression of different TF i.e. Hox genes that specify the AP axis
• Hox genes feedback to give a new local zones of RA in these regions
• RA in combination with the Hox code specifies the expression of different Tbx TF that generate limb identity

Question 11

What does Tbx gene expression set up?

Answer 11

Zones of FGF expression

Question 12

What is FGF expression important for?

Answer 12

Proliferation

Question 13

Where is the forelimb generated?

Answer 13

In the lateral plate mesoderm (outside of the paraxial mesoderm, next to somites 18-20

Question 14

Where is the hindlimb generated?

Answer 14

The hindlimb is also generated from lateral plate mesoderm, but in somites 26-28

Question 15

What happens when a bead is soaked in FGF and transplanted to an ectopic site?

Answer 15

• A bead soaked in FGF, mimics the action of the limb field to induce ectopic development of limbs i.e. it resulted in a 3rd limb, showing the outgrowth of a limb in a responsive field is promoted by FGF.

Question 16

Describe the model for limb bud formation

Answer 16

• There is an initial burst of FGF8 in intermediate mesoderm.
• Then, Tbx TF (4/5) have be induced. This induces TF that act cell-autonomously on cells to upregulate the FGF10 encoding gene.
• The entire lateral plate mesoderm, that sits under the surface ectoderm, expresses and translates FGF10 in the mesoderm.
• FGF10 is a secreted molecule that acts on adjacent AER to induce these cells to upregulate a 2nd TF called FGF8.
• Domains of FGF10 expression are restricted along AP by Wnt2b.
• Wnt3a is induced in the limb field
• FGF8 acts on the progress zone

Question 17

What is the signalling interaction between FGF10/8

Answer 17

This is a reciprocal signalling interaction as FGF10 signals to surface ectoderm so it starts to upregulate FGF8 that signals back to lateral plate mesoderm. This slowly restricts cells and gives more specification over time.

Question 18

Describe the progress zone

Answer 18

• Made up primarily of mesenchymal cells

- Expresses FGF10

Question 19

Describe the apical ectodermal ridge

Answer 19

• Expresses FGF8

Question 20

Describe the Zone of Polarising Activity

Answer 20

• Expresses Shh (morphogen)

- Found posterior of the limb bud

Question 21

Describe the progress zone model

Answer 21

Suggests the AER is driving outgrowth of the limb
AER then acts on the progress zone. The longer the progenitor cell sits as a proliferating cell in the PZ, the more distal the fate. The TIMING of when a cell differentiates is key to its fate

Question 22

Describe the two signal model

Answer 22

Initial progenitors are present for all the skeletal derivatives. Under influence of Hox code a domain of RA forms in flank where the limb bud originates. Over time FGF8 is induced in the AER. Sets up 2 opposing gradients of the 2 antagonistic signals (RA/FGF8). Cells encountering increased RA become more proximal.

Question 23

Where is this interpretation of positional information conserved?

Answer 23

Flies

Question 24

What is the fly homologue of Hoxd13

Answer 24

Distalless

Question 25

What is the fly homologue of Meis

Answer 25

Homothorax

Question 26

TYPICAL EXAM Q: What is the evidence Shh mediates the activity in the ZPA?

Answer 26

• Action of ZPA is mimicked by soaking a bead in Shh or forcing the expression in cells which don’t usually express it.
• Transfected shh-expressing virus into an infectable strain of chick embryo fibroblast cells.
• Pellet cells were selected that could secrete the Shh protein.
• Implant cells ectopically into the A part of limb bud, so the host embryo has own ZPA and ectopic ZPA.
• Also get mirror image duplication.

Question 27

What is Wolpert’s French Flag model for patterning

Answer 27

Shh is expressed in cells of ZPA but the protein is secreted to establish a morphogen gradient so cells furthest from the ZPA (lowest conc of SHh) form digit 2, intermediate levels form digit 3 and those closest to the ZPA (highest conc) form digit 4

Question 28

Describe how the proximo-distal and antero-posterior patterning is coordinated

Answer 28

• Shh from the ZPA diffuses P to A. Fgf4 from the AER diffuses distal to proximal
• Fgf8 expression in AER is established by Fgf10 from mesoderm.
• Yet, when ZPA is established later, Shh operates to maintain expression of Fgf8 in AER.
• Fgf8 maintains expression of Shh in ZPA
• i.e. ZPA/AER work together to maintain right patterning and outgrowth of AP/proximo-distal axes