L15 - Vertebrate Limb Formation

Question 1

What are limb buds?

Answer 1

First appear as protrusions from the flank at precise positions along the AP axis

Question 2

How can an ectopic limb be formed?

Answer 2

Determination of the limb field is an intrinsic property to the lateral mesoderm
Can take a piece of mesoderm from prospective field
Transplant to an area of the flank not normally forming a limb
A limb now forms here

Question 3

How were T-box transcription factors identified?

Answer 3

In-situ hybridisation

Question 4

Which two T-box transcription factors specify limb identity?

Answer 4

Restricted in their expression pattern to the limb fields
Either expressed in the forelimb or hindlimb bud
- Tbx5 – forelimb
- Tbx4 – hindlimb

Question 5

How does Tbx5 influence Tbx4?

Answer 5

Tbx5 represses expression of Tbx4

- Forced expression of Tbx5 in hindlimb region switched of Tbx4

Question 6

What did experiments with T-box transcription factors in the chick show?

Answer 6

Gain of function studies
Force expression of these genes in their opposite locations
- Reversal of identity
Mouse studies do not confirm this
- Loss of function of Tbx4 – hindlimb still forms

Question 7

Where is Ptx1 expressed?

Answer 7

Uniquely expressed in the hindlimb

Drives limb identity

Question 8

What happens in a Ptx1 knockout?

Answer 8

Lose tbx4 expression and hindlimb formation

Question 9

What is the series of proteins involved in limb formation?

Answer 9

Hox proteins –> Retinoic acid –> Tbx transcription factors –> Fgf10 (mesoderm) –> Fgf8 (ectoderm)

Question 10

Limb formation method

Answer 10

1. Hox proteins along A-P axis drive positional identity of limb fields
2. Forms Hindlimb or forelimb expressing different Tbx transcription factors
3. These drive expression of Fgf10
4. This drives expression of Fgf8
   o Activity of Fgf8 also generated by intermediate mesoderm
   o Initially broad domain of expression
   o Wnt signalling restricts this
5. Relay of information from intermediate mesoderm –> lateral mesoderm –> ectoderm

Question 11

What does Hox6 control?

Answer 11

Marks transition between cervical and thoracic vertebrate

Marks where cervical and thoracic vertebrate will form

Question 12

What happens if you replace limb field cells with beads soaked in FGF4/FGF8?

Answer 12

Ectopic limb

Question 13

What are the 3 zones in the early limb bud?

Answer 13

Apical ectodermal region
Progress zone
Polarising zone

Question 14

What s the apical ectodermal region?

Answer 14

Thickening of ectoderm overlying the mesoderm

Rich in Fgfs

Question 15

What is the progress zone?

Answer 15

Mesenchymal cells

Lies just behind apical ectodermal ridge

Question 16

What is the polarising zone?

Answer 16

Expresses Shh

Most posterior

Question 17

How was positional information along the P-D axis shown through experiments?

Answer 17

Removes ectoderm from developing embryo at different time points

• At 3 days – only most proximal wing bone forms
• At 3.5 days – more lateral skeleton development
• At 4 days – all of lateral skeleton formed – just no digits

Question 18

What were the conclusions from the experiment where ectoderm was removed from the developing embryo at different time points?

Answer 18

Apical ectodermal region is necessary for the progress zone (limb growth)
More distal skeletal elements are specified as the limb grows

Question 19

What are the two models for P-D patterning of the vertebrate limb?

Answer 19

Progress zone

Two signal model

Question 20

What is the progress zone model?

Answer 20

AER was driving outgrowth of the limb by acting on the progress zone
Promotes proliferation of the cells
The more the cells divided in the progress zone the more distal structures formed
Now known to not be correct after using fate mapping techniques
- Early on in developing limb field you already have progenitors that can contribute to all the skeletal elements present in the future limb

Question 21

What is the two signal model?

Answer 21

Initially have progenitor cells for all skeletal elements of the limb
These cells will adopt a specific cell fate depending on the signal they receive
- Will receive antagonistic signals from P-D region
- According to their position along the gradient they will either become a proximal or distal element

Question 22

In vertebrates what molecules are expressed distally?

Answer 22

Fgf

Wnt

Question 23

In vertebrates what molecule is expressed proximally?

Answer 23

Retinoic acid

Question 24

How is the P-D axis patterned?

Answer 24

Fgf/Wnt and retinoic acid together create antagonistic gradients causing cells to adopt different fates

Question 25

How is the Fgf/Wnt and retinoic acid gradient translated intrinsically?

Answer 25

Retinoic acid drives expression of Meis genes FGFs drives expression of - Hox13 gene – most distal - Hox11 – most proximal

Question 26

Interpretation of positional information is conserved in flies and vertebrates along the P-D axis

Answer 26

In drosophila Patterning controlled by Distalless and homothorax Distalless is homologue of Hox13 Homothorax is homologue of Meis

Question 27

What controls A-P patterning?

Answer 27

Shh signals from the ZPA

Question 28

What experiments when done to look at the signals created by the ZPA?

Answer 28

Anterior graft of ZPA creates a mirror image duplication of autopods – due to reversed morphogen gradient If graft performed early, there is also duplication of ulna Beads soaked in Shh also produced this mirror image effect

Question 29

What were the conclusions to the experiments done on the ZPA?

Answer 29

ZPA drives patterning of cells along A-P entirely due to action of Shh Led to Wolperts French flag model - A-P patterning controlled by a morphogen gradient - High concentrations – drives expression of digit 4 - Closest to ZPA - Low concentrations – drives expression of digit 2

Question 30

What is the phenotype of Shh mutants?

Answer 30

Phenotype of Shh mutant limb is consistent with the morphogen gradient hypothesis Absence of Shh - Complete loss of distal most skeletal elements - Loss of identity of the zeugopods - Truncation of limb

Question 31

How is their coordination between P-D and A-P patterning?

Answer 31

Through reciprocal control of events - Signalling molecules in the AER responsible for elongation of limbs (P-D axis) are required to maintain expression of Shh in the ZPA - The ZPA is required to maintain FGF expression in the AER

Question 32

What three genes are involved in D-V patterning?

Answer 32

Wnt7a – dorsal Engrailed1 – ventral Lmx1b

Question 33

What experiments where done in the chick to show what patterned the D-V axis?

Answer 33

Ectoderm covering the dorsal aspect of the limb differs from that covering the ventral aspect Differential expression between - Wnt7a – dorsal - Engrailed1 – ventral Lmx1b transcription factor expression divides the limb along the D-V axis

Question 34

What does a conditional knockout of Lmbx1 cause?

Answer 34

Ventralisation of the limb

Question 35

Model for control of D-V patterning

Answer 35

1. BMP signalling in the ventral dives expression of Engrailed1 in ventral limb 2 This represses the expression of Wnt7a 4. Wnt7a becomes restricted to the dorsal ectoderm 5. Drives expression of Lmx1b in dorsal part of the limb 6. This transcription factor is vital in dorsal patterning of the limb

Question 36

P-D axis formation summary

Answer 36

AER secreted FGF4/8 and drives limb outgrowth | Two models – progress zone and two signal

Question 37

A-P axis formation summary

Answer 37

ZPA secretes Shh which forms a gradient along the AP acis Cell specification determined according to concentration of Shh – digits and radius/ulna P-D and A-P axis are coordinated through reciprocal control between AER and ZPA

Question 38

D-V axis formation summary

Answer 38

Dorsal and ventral ectoderm (Wnt7a and BMP) control Lmx1b which drives dorsal identity