Co-authors (left to right): Hongkai Ji, Steven Vokes, Andrew McMahon and Wing H. Wong
The vertebrate limb has been one of the central models for understanding the problem of complex pattern formation. The critical determinant is Sonic hedgehog, a posterior mesenchymal signal that is required for the establishment of the appropriate number and identity of digits. The types of analyses that have been performed to address Shh function have largely centered on altering time, positioning and duration of Shh action and looking at the final consequences in terms of digit output. What has been absent is a real understanding of the primary targets of this pathway, an understanding that is essential to forward the field from its current abstract, black-box status to one firmly grounded on the realities of the Shh-mediated transcriptional response. In this study we attempt to bring the Shh pathway to this level of understanding by exploring the direct regulatory output of this network using a whole-genome chromatin immunoprecipitation approach with the key transcriptional effector Gli3; to our knowledge the first such analysis on this scale in a mammalian developmental system.
There are several important points to note about the data. First, the targets could not be predicted by current in silico approaches or by ChIP analysis alone, but require the intersection of ChIP binding regions and transcriptional profiling to provide a robust set of predictions. Second, Gli target sites are very frequently at considerable distances from the transcriptional start sites of their target genes (greater than 20 kilobases). This contrasts strikingly with developmental models in C. elegans and Drosophila and depicts one of the challenges faced with understanding the regulatory circuitry in mammals. Finally, the data provide important insight on the targets and regulatory processes at play in limb outgrowth and patterning. For example, contrary to popular models where Shh regulates limb pattern exclusively through Gli3 de-repression, direct Gli activation plays a critical role in the limb. This is exemplified by our analysis of Gremlin regulation. Gremlin is a key factor in limb outgrowth and it acts in a relay from Shh to the apical ectoderm to maintain production of signals that control outgrowth of the limb. Our data indicate that Gremlin is likely regulated by dual inputs: one from a Shh-mediated Gli activator response via a cis-element over 100 kb from the Gremlin transcriptional start site; the other we argue is likely a more general BMP input.