The Role of Fgf Signaling and Limb Outgrowth
Mariani FV, Ahn CP, Martin GR. 2008 -Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning. Nature. 453(7193):401-5.
Mariani FV, Martin GR. 2003. Deciphering skeletal patterning: clues from the limb. Nature. 423(6937):319-25
Briefly tell us a bit about yourself, your career paths over the years, and specifically what led you to begin working on limb development. There were many student-generated questions about various aspects of the several models proposed for limb bud outgrowth. To frame today’s discussion can you very briefly describe the main position of these different models as you see them?
What is the difference between permissive and instructive AER-FGF signaling for limb bud patterning?
From the data, loss of Fgf8 seems to have a detrimental effect on skeletal patterning, while loss of the other AER-FGFs not as much. What are the advantages or disadvantages of the functionally redundant AER-FGFs (Fgf4, Fgf9 and Fgf17) in the positive feedback loop between the AER and the patterning center in posterior limb bud mesenchyme?
In previous work, you’ve found that skeletal development fails to happen in Fgf4;Fgf8 double KOs, and Fgf9 and Fgf17 aren’t able to rescue it. What role do Fgf9 and Ffg17 play independent from the other FGFs? Have you researched what happens in Fgf9 or Fgf17 single knockouts?
I’m curious as to why the stylopod decreased in size for the F8;F4-DKO;F9-/+ mutants (Fig. 4), according to the model you propose. If the development of the stylopod is regulated by proximal signals, which oppose FGFs (distal signals), then why doesn’t it remain the same size? If distal signaling is reduced, why is the proximal skeletal element affected if limb segments are specified at early stages?
Based on the two-signal model of limb specification, why isn’t there a middle zeugopod domain in the F8;4-DKO;F9-/+ forelimbs? Shouldn’t there be some FGF and retinoic acid overlap if there is enough FGF signal to specify autopod development? And, what data holds other experimenters from accepting your model as feasible after this paper demonstrates that AER signals are instructive for a dorsal fate rather than time spent in the proximal zone?
Considering the model proposed in the final figure in the 2008 paper, do you think it would be a worthwhile experiment to find a way to reverse the signals from the proximal and distal areas (or physically flip the specified areas of the limb bud at E 10.5) and see how the limb develops? What would you expect to see when running an experiment like this? Would this help to further shed light on which of the two models are more likely to be correct?
You explain that when AER-FGF signaling is decreased, distal signaling is proportionally decreased, causing more stylopod or proximal development. However, since you don’t get an extra long stylopod in this case, some mechanism is inducing apoptosis in these cells. Wouldn’t this then suggest a temporal regulation of cell fate determination, rather than cell specification before the change in AER-FGF signaling?
Questions of death. The article mentions that limb buds with more AER-FGF signaling have a cell death domain that occupies a smaller percentage of the limb bud volume. In F8;4-DKO;F9-/+ mutants, mesenchymal cell death was detected in a proximal dorsal region rather than distally. Why do those cells die rather than develop into proximal skeletal elements? Are Fgf8 and Fgf4 actively repressing genes that control cell-death pathways?
How applicable is this analysis for the hindlimb? Has your lab further tested whether cell proliferation of the hindlimb mesenchyme is reduced in later stages (>35 somites) in the double and triple knockout mutanst? If not, can you hypothesize as to what the result might be?
In the Fgf8;4 DKO; Fgf9 -/- mutant, nail development seems to occur, even though the limb that is most distal is not necessarily a digit. You have suggested that Fgfs are responsible for distal cell fate so are nails not included in this category? What is responsible for nail formation?
Currently in your opinion what are the most pressing questions in the field of limb develop, and what steps is your lab taking to address those questions?