Molecular Mechanisms of Patterning the Limb Proximodistal Axis and Quantitative Studies Addressing its Scalability over Five-Fold Changes in Size.
Final Report Abstract
In this project we were able to use heterochronic transplantation showing that onset of HOXA13 appears to be on some kind of cell autonomous timer, as transplantation of young cells into older blastemas did not accelerate HOXA13 expression. As part of the studies, we also tested whether FGF8 and Retinoic acid have inductive effects on expression of proximo-distal identity genes including HOXA13 and MEIS. These factors were chosen based on the prevalent two-signal model for vertebrate limb bud development. Consistent with a cell autonomous timer controling HOXA13 onset, functional testing of the FGF and RA pathway using expression of dominant negative receptors did not yet reveal a strong determinative role of these factors in the induction of the proximal-distal identity genes. Due to the slightly unexpected negative result on the effect of RA pathway perturbation on MEIS expression, we performed some experiments beyond the original scope of the grant to gain further insight into the control of MEIS expression in regeneration. We used heterotopic transplantation to understand to what extent limb cells have a pre-defined set point for MEIS activation. We also used molecular profiling to start to gain an insight on potential modulators. Secondly, given what appears to be cell intrinsic regulators of proximal-distal cell identity, we took advantage of the possibility to make CRISPR-mediated mutants to test the role of EZH2 in this patterning system. Finally to gain insight into the scaling system in different sized animals, we screened the expression of FGF8 and MEIS pathway components and could show that indeed the sizes of these domains scale in different sized blastemas. Due to some of the unexpected results, and the generation time required to make transgenic axolotls, we are still in process of pursuing the results for final publication.
