LIMB-TYPE ID · Identification of candidate limb type identity determining genes

Identification of candidate limb type identity determining genes

It is now widely appreciated that the same signalling pathways contribute to the development of many distinct structures of the body. This observation raises the fundamental questions in developmental biology of how discrete structures emerge from identical cell population using identical signalling cascades. An ideal model to address this problem is the vertebrate limb. Vertebrates have two pairs of homologous appendages, forelimbs and hindlimbs. In most respects, the genetic networks controlling limb development act equivalently in both appendages. However, in order to adapt to their distinct functions, the forelimbs and hindlimbs have developed specific structural elements. Progenitors of the various structures of the forelimb and hindlimb must therefore respond differently to common signalling inputs. In the past decade significant breakthroughs have been made in our understanding of limb patterning; however the molecular processes leading to limb-type morphology determination remains unclear. So far, the only mouse gene that has been implicated in limbtype specification is Pitx1, a homeobox transcription factor. The Pitx1 gene constitutes therefore an important entry point to further describe these processes. Pitx1, expressed in the hindlimb bud mesenchyme, is required for the formation of hindlimb characteristics and produces hindlimb-like morphologies when misexpressed in forelimbs. Pitx1 is also necessary for normal expression of Tbx4, a transcription factor required for normal hindlimb development. Despite the importance of this protein in these processes, little is known about its mechanism of action. Using a transgenic gene replacement strategy in a Pitx1 mutant mouse, we have uncoupled two discrete functions of Pitx1. We have shown that, firstly, this protein influences hindlimb outgrowth by regulating Tbx4 expression levels and that, subsequently, it shapes hindlimb bone and soft tissue morphology independently of Tbx4. We have provided the first description of how Pitx1 sculpts the forming hindlimb skeleton by localised modulation of the growth rate of discrete elements (Duboc V. and Logan M. P. O.; 2011). The transcriptional targets of Pitx1 remain unknown. In light of this analysis, I have been performing a series of microarray that allowed us to identify Tbx4'outgrowth'targets and Pitx1'Morphology'targets by comparing the transcriptomes of Pitx1 -/- hindlimbs to Tbx4 rescues of the Pitx1 -/- phenotype and wild type hindlimb transcriptomes, subject of another research article. In addition, I also initiated a longitudinal analysis of the transcriptomes using of wild type forelimb and hindlimbs RNAseq.comparing the transcriptional profiles of forelimbs and hindlimbs over an embryonic time-course encompassing initiation of outgrowth, patterning and early differentiation stages, allows us not only to identify additional candidate "limb-type modifiers" but mainly to compare the transcriptional profiles dynamics between forelimb and hindlimb and their common gene regulatory networks to identify limb type signatures. This data set will also serve as a reference database for future expression analysis in different mutant contexts. These results are the subject of upcoming research articles.

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