L15 - Vertebrate Limb Formation Flashcards
What are limb buds?
First appear as protrusions from the flank at precise positions along the AP axis
How can an ectopic limb be formed?
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
How were T-box transcription factors identified?
In-situ hybridisation
Which two T-box transcription factors specify limb identity?
Restricted in their expression pattern to the limb fields
Either expressed in the forelimb or hindlimb bud
- Tbx5 – forelimb
- Tbx4 – hindlimb
How does Tbx5 influence Tbx4?
Tbx5 represses expression of Tbx4
- Forced expression of Tbx5 in hindlimb region switched of Tbx4
What did experiments with T-box transcription factors in the chick show?
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
Where is Ptx1 expressed?
Uniquely expressed in the hindlimb
Drives limb identity
What happens in a Ptx1 knockout?
Lose tbx4 expression and hindlimb formation
What is the series of proteins involved in limb formation?
Hox proteins –> Retinoic acid –> Tbx transcription factors –> Fgf10 (mesoderm) –> Fgf8 (ectoderm)
Limb formation method
- Hox proteins along A-P axis drive positional identity of limb fields
- Forms Hindlimb or forelimb expressing different Tbx transcription factors
- These drive expression of Fgf10
- 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 - Relay of information from intermediate mesoderm –> lateral mesoderm –> ectoderm
What does Hox6 control?
Marks transition between cervical and thoracic vertebrate
Marks where cervical and thoracic vertebrate will form
What happens if you replace limb field cells with beads soaked in FGF4/FGF8?
Ectopic limb
What are the 3 zones in the early limb bud?
Apical ectodermal region
Progress zone
Polarising zone
What s the apical ectodermal region?
Thickening of ectoderm overlying the mesoderm
Rich in Fgfs
What is the progress zone?
Mesenchymal cells
Lies just behind apical ectodermal ridge
What is the polarising zone?
Expresses Shh
Most posterior
How was positional information along the P-D axis shown through experiments?
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
What were the conclusions from the experiment where ectoderm was removed from the developing embryo at different time points?
Apical ectodermal region is necessary for the progress zone (limb growth)
More distal skeletal elements are specified as the limb grows
What are the two models for P-D patterning of the vertebrate limb?
Progress zone
Two signal model
What is the progress zone model?
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
What is the two signal model?
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
In vertebrates what molecules are expressed distally?
Fgf
Wnt
In vertebrates what molecule is expressed proximally?
Retinoic acid
How is the P-D axis patterned?
Fgf/Wnt and retinoic acid together create antagonistic gradients causing cells to adopt different fates