L15 - Vertebrate Limb Formation Flashcards

1
Q

What are limb buds?

A

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

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2
Q

How can an ectopic limb be formed?

A

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

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3
Q

How were T-box transcription factors identified?

A

In-situ hybridisation

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4
Q

Which two T-box transcription factors specify limb identity?

A

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

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5
Q

How does Tbx5 influence Tbx4?

A

Tbx5 represses expression of Tbx4

- Forced expression of Tbx5 in hindlimb region switched of Tbx4

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6
Q

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

A

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

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7
Q

Where is Ptx1 expressed?

A

Uniquely expressed in the hindlimb

Drives limb identity

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8
Q

What happens in a Ptx1 knockout?

A

Lose tbx4 expression and hindlimb formation

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9
Q

What is the series of proteins involved in limb formation?

A

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

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10
Q

Limb formation method

A
  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
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11
Q

What does Hox6 control?

A

Marks transition between cervical and thoracic vertebrate

Marks where cervical and thoracic vertebrate will form

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12
Q

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

A

Ectopic limb

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13
Q

What are the 3 zones in the early limb bud?

A

Apical ectodermal region
Progress zone
Polarising zone

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14
Q

What s the apical ectodermal region?

A

Thickening of ectoderm overlying the mesoderm

Rich in Fgfs

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15
Q

What is the progress zone?

A

Mesenchymal cells

Lies just behind apical ectodermal ridge

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16
Q

What is the polarising zone?

A

Expresses Shh

Most posterior

17
Q

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

A

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
18
Q

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

A

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

19
Q

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

A

Progress zone

Two signal model

20
Q

What is the progress zone model?

A

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

21
Q

What is the two signal model?

A

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

22
Q

In vertebrates what molecules are expressed distally?

23
Q

In vertebrates what molecule is expressed proximally?

A

Retinoic acid

24
Q

How is the P-D axis patterned?

A

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

25
How is the Fgf/Wnt and retinoic acid gradient translated intrinsically?
Retinoic acid drives expression of Meis genes FGFs drives expression of - Hox13 gene – most distal - Hox11 – most proximal
26
Interpretation of positional information is conserved in flies and vertebrates along the P-D axis
In drosophila Patterning controlled by Distalless and homothorax Distalless is homologue of Hox13 Homothorax is homologue of Meis
27
What controls A-P patterning?
Shh signals from the ZPA
28
What experiments when done to look at the signals created by the ZPA?
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
29
What were the conclusions to the experiments done on the ZPA?
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
30
What is the phenotype of Shh mutants?
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
31
How is their coordination between P-D and A-P patterning?
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
32
What three genes are involved in D-V patterning?
Wnt7a – dorsal Engrailed1 – ventral Lmx1b
33
What experiments where done in the chick to show what patterned the D-V axis?
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
34
What does a conditional knockout of Lmbx1 cause?
Ventralisation of the limb
35
Model for control of D-V patterning
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
36
P-D axis formation summary
AER secreted FGF4/8 and drives limb outgrowth | Two models – progress zone and two signal
37
A-P axis formation summary
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
38
D-V axis formation summary
Dorsal and ventral ectoderm (Wnt7a and BMP) control Lmx1b which drives dorsal identity