Lecture 15- Vertebrate limb formation Flashcards
Define pattern formation
Coordinated differentiation and organisation of cells within the body in order to form structures that are specific size, shape and position within and organism
Define positional information
Cells in a specific space within the developing embryo have intrinsic information that indicates that they must carry out a specific programme which is different to cells in different positions
What are the 3 axes of the vertebrate limb?
- The P/D axis: the shoulder/humerus is proximal, the digits is distal
- The A/P axis: the thumb is anterior, the pinky digit is posterior
- The D/V axis: the palm is dorsal, the back of the hand is ventral
What are the 3 skeletal elements of the vertebrate limb and what bones to they compose of?
- Stylopod- humerus
- Zeugopod- ulna and radius
- Autopod- carpals and digits
Which axis cannot be defined through the organisation of bones but instead defined through an alternative method?
The D/V axis cannot be defines through the organisation of the bones however the specific features of the epidermis vary on the dorsal and ventral sides
Describe the first appearance of the limb bud
Protrusions from the flank at precise positions along the AP axis of the embryo
What dictates the position of the limb fields and give an example?
Hox code
Anterior boundary of Hoxc6 specifies the forelimb field
What 2 transcription factors specify limb identity?
Tbx4 and Tbx5
Where is Tbx4 and Tbx5 expressed?
Tbx4 expressed in the hindlimb
Tbx5 expressed in the forelimb
What drives the identity of Tbx4 and the hindlimb?
Pitx1
What represses Tbx4?
Tbx5
Describe 1 gain of function study for Tbx4/5
If you prepared a virus that will overexpress Tbx4 and inject the virus so that Tbx4 is expressed throughout the lateral mesoderm, the embryo will form a leg in the forelimb area. The opposite is observed when Tbx5 is overexpressed
Describe 1 loss of function study for Tbx4/5
By blocking expression of Tbx5 (e.g through mouse KO), forelimb formation is lost and the hindlimb formation is not effected. Indicates that Tbx4 is sufficient to drive hindlimb formation and Tbx5 is required for forelimb bud formation
What is observed the Pitx1 is KO or overexpressed?
KO: loose Tbx4 expression and hindlimb identifty
Overexpressed: Tbx4 expression is driven and hindlimb forms despite the region
Describe the model for imitation of limb bud formation from Hox proteins to FGFs
Hox protein - RA - Tbx - FGF10 (mesoderm) - FGF8 (ectoderm)
What regulates the expression of Tbx4/5?
RA
Outline an experiment in which a limb is ectopically formed
FGF soaked bead loaded into the lateral mesoderm of the developing embryos leads to the ectopic formation of a limb
What in the link between FGF and Tax gene expression in the developing limb?
Tbx4/5 drive the expression of FGF10 in the lateral mesoderm which drives the expression of FGF8 in the ectoderm
Which 2 ways were embryos analysed after the ectopic expression of FGF10 in the limb?
- insitu hybridisation
2. Visualise the morphology of the developing limb
How does the type of limb formed change depending on the location of the ectopic FGF10 soaked bead>
- If the bead was implanted closer to the hindlimb, then hindlimb formed
- If the bead was implanted closer to the forelimb, then forelimb formed
- If the bead was implanted precisely between the two, then the developing limb partially expressed both Tbx4 (posteriorly) and Tbx5 (anteriorly). The ectopic limb developed morphologically features that were chimeric and resembled both a hindlimb and forelimb
What conclusions were made from ectopic implantation of FGF10 soaked bead in the developing limb
Concluded that FGF10 is important for driving the expression of Tbx4/5 in the limb
Outline the molecular expression that enabled forelimb formation initiation
- RA and FGF10s important for expression of Tbx5
- Wnt2b acts to restrict the expression pattern solely to the limb field
- FGF10 in the lateral mesoderm establishes features within the limb field which are important for the later growth
- FGF10 induces in the overlying ectoderm the expression of Wnt3a which in turn induces the expression of FGF8
- Wnt3a and FGF8 together within the ectoderm define the AER/progress zone
Outline the molecular expression that enabled hindlimb formation initiation
- Pitx1 and Tbx4 together are important for the formation and identify of the hindlimb limb field
- FGF10 and Tbx4 also lead to Wnt8c which acts to restrict the expression pattern solely to the limb field
- FGF10 induces in the overlying ectoderm the expression of Wnt3a which induces the expression of FGF8 to define the AER/progress zone
What are the 3 domains which interact with each other to drive the growth and patterning of the limb?
- Apical Ectodermal Ridge (AER): will become specified under the action of FGF10. Area forms through the thickening of ectoderm and expresses a high number of FGFs
- Progress zone: made of mesenchymal cells that are actively dividing and promoting the growth of the limb and located behind the AER
- Zone of Polarising Activity (ZPA): region expresses high levels of Shh
Experimentally removing the AER confirms the AER is necessary for what?
The progress zone and limb growth
What is the resulting phenotype when the AER is removed at 3, 3.5 and 4 days?
- If the AER was removed at 3 days, the resulting embryo developed stylopod, some zeugopod but no autopod
- If the AER was removed at 3.5 days, the resulting embryo limb developed stylopod, zeugopod but again, no autopod
- If the AER was removed at 4 days, the resulting limb developed stylopod, zeugopod and some autopod
What are the 2 models for P/D patterning of the vertebrate limb?
Model 1: progress zone model of proximal-distal patterning
Model 2: two-signal model of proximal-distal patterning
Describe the progress zone model of P/D patterning
- The AER was driving the outgrowth of the limb by acting on the progress zone by promoting the proliferation of the cell
- Cells were adopting a specific cell fate according to the time spent dividing in the progress zone
- The time spent in the progress zone directly correlates with how distal the elements forming were
- The more proximal elements were specified early, and the cells that spent longer in the progress zone would develop into more distal elements
Describe the two signal model of P/D patterning
- Two antagonising gradients that are established early which act together to specify different regions within the developing limb and ultimately results in the formation of different regions that are patterned
- The cells position along the opposing signals gradient will determine there proximal or distal fate
- RA and FGF were the two signals antagonising each other
Describe the RA and FGF gradients in the limb bud
- High levels of RA in the proximal part of the limb and decreases distally
- High levels of FGF in the distal part of the limb and decreases proximally
What genes are expressed where due to FGF/RA in the limb bud?
- Proximally Meis genes are activated by high RA and are required to form the stylopod
- Distally, Hox13 genes are activated by high FGFs and required to form the autopod
- In the middle (neither proximal or distal with mixed levels of RA and FGFs), Hox11 is activated and required to form the zeugopod
What can be observed and concluded from implanting Shh beads/Shh expressing cells/ZPA grafts?
Mirror image duplication of the limb
Concludes that ZPA which drives A/P patterning canoe entirely due to Shh activity
What zones pattern the A/P and P/D axis of the limb?
A/P = ZPA D/P = AER and progress zone
How does Shh concentrations control the cell types specified?
- In the ZPA, there are high levels of Shh which decrease as you move further away
- Therefore the most posterior digits are specified close to the ZPA where Shh is at high concentrations
- Anterior digits are specified further away from the ZPA where Shh is at lower concentrations
What is observed in a Shh mutant limb?
A complete loss of the distal most skeletal elements but the most proximal elements develop as can develop without Shh present
Also, loss of identity of the zeugopods
Describe the feedback loops established between the AER and ZPA that allows the limb to develop along both the A/P and D/P axis
ZPA is important for maintaining FGF4/8 expression in the AER
FGF8 in the AER is important for maintain Shh signalling in the ZPA
What 3 genes are essential for D/V patterning of the limb and where are they expressed?
- Wnt7a is specifically expressed in the dorsal ectoderm
- En1 is specifically expressed in the ventral ectoderm
- Lmx1b is specifically expressed in the mesenchyme of the dorsal limb
What is observed and concluded when Lmx1b is knocked out in the limb mesenchyme?
- Causes the ventilation of the limb and the loss of dorsal features
- Confirming that Lmx1b is required for specifying the dorsal pattern of the limb and in the absence the limb mesenchyme adopts a alternative ventral fate
- The ventral identify is therefore the default pathway
What is the proposed model for controlling the D/V patterning of the limb?
- BMP signalling occurs in the ventral part of the limb which drives the ventral expression of En1
- En1 expression inhibits and restricts the expression of Wnt7a to the limbs dorsal ectoderm
- Wnt7a drives the expression of Lmx1b which is only expressed in the dorsal half of the limb
What is observed if the AER is removed at an early stage of limb development?
A limb without a humerus/radius/ulna etc
Limb development ceases
What is observed if an additional AER is grafted anterior to the existing one at an early stage of limb development?
A limb with mirror image duplication of digits as the extra AER FGFs induce the formation of another ZPA
What is observed if a leg mesenchyme is grafted in place of the limb mesenchyme at an early stage of limb development?
A limb with characteristics of a leg
What is observed if a non-limb mesenchyme is grafted in place of the wing mesenchyme at an early stage of limb development?
A truncated limb
What is observed if the AER was replaced by FGF bead at an early stage of development?
A normal limb develops
