Limb morphogenesis, from a small mesenchymal bulge to a highly reproducible and functional final structure, requires integration of multiple signalling pathways.
The transcription factor Gli3, acting as the primary mediator in the limb of the secreted morphogen Sonic hedgehog (Shh), is central to specifying digit number and identity. Loss of Gli3 function in mouse results in the polydactylous mutant extra-toes (Gli3Xt/Xt), while aberrant Gli3 expression in humans results in a spectrum of developmental disorders all exhibiting limb defects. From early in limb development, Gli3 acts to define boundaries of gene expression, establishing asymmetric patterns required to provide positional information. Analysis of Gli3 dependent gene expression however has been limited to a small number of signalling molecules previously implicated in limb development. The advent of large scale gene expression analysis has allowed us to perform a more complete and unbiased investigation into the transcriptional consequences
of loss of Gli3, with the ultimate goal of identifying novel molecules missing from the known hierarchies governing limb development and Polydactyly.
Using microarray approaches, a direct comparison was made between RNA isolated from Gli3Xt/Xt and wild-type anterior limb buds at both embryonic day (E)11.5 and El2.5. As a result, we have identified 61 transcripts as potentially regulated by Gli3 at both embryonic stages. A strong downregulation of Gli3 expression was identified in Gli3Xt/Xt limbs, validating our experimental approach. The remaining 60 transcripts have not been characterised in the Gli3Xt/Xt
mutant limb bud to date, though many have been implicated in Shh signalling. We have validated altered expression of 16 transcripts within the Gli3Xt/Xt limb. This was achieved primarily by whole mount in situ hybridisation analysis which allows spatial evaluation of gene expression and underpins our ability to rationalise changes in gene expression in terms of developmental processes.
This work has led to the finding that the Notch signalling cascade may be a downstream effector of Gli3 in the limb, as Gli3 regulates spatial restriction of the Notch ligand, Jagged1. Pax9 expression in the anterior limb bud was identified as almost entirely dependent on Gli3, which is in contrast to redundant modes of
Pax9 regulation observed in other embryonic contexts. We have also identified a delay in chondrogenesis specifically in the anterior Gli3Xt/Xt limb bud, which we believe may be central to phenotypic alterations observed in these mice. In addition to known genes, we have validated a number of completely uncharacterised transcripts as dependent on Gli3 expression in the vertebrate limb. In this way, we have expanded the repertoire of limb markers which may help elucidate wider aspects of limb development.