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Transcriptional and cis-regulatory mechanisms underlie the process of cellular specification during embryonic development. Here, initiating signals tell undifferentiated cells their fates and positions within an organ. In many cases these initiating signals are known, but it is not understood how these signals are integrated into a quantitative transcriptional output that is interpreted by the cells. We seek to understand how cells take stock of all of the inductive signals they receive from their neighbors and translate this into a genetic network that specifies their fate and position.
To address this goal, we use the mouse embryo, focusing on the transcriptional regulation of limb size during embryonic development. In this process, a limb bud consisting of a small population of mesenchyme encased in an outer layer of ectoderm, is transformed into the various skeletal elements of the vertebrate limb. Limb outgrowth is driven by the interplay between two organizing centers that collectively regulate the size and polarity of the limb elements. The pathways regulating these processes are relatively well characterized at the signaling level, but are sparsely understood at the transcriptional level, the ultimate level of signal integration.
As an initial step towards characterizing transcriptional networks, we have focused on the Sonic hedgehog (Shh) signaling pathway in the mouse embryo using a combination of genomic and genetic techniques. This pathway is critical for multiple steps in embryonic development; mutations in the pathway cause a spectrum of birth defects that include craniofacial defects and polydactyly (extra digits). Importantly, all transcriptional activity is mediated by the Gli proteins (Gli1-3).
In order to determine direct Gli target genes, we utilized techniques that, for the first time, allow whole-genome ChIP-on-chip analysis of mouse embryos in a tissue specific fashion. These were collaborative studies with Wing Wong and Hongkai Ji. This involved the generation of mice containing an inducible, epitope-tagged Gli3. These experiments showed that the limb bud is bound by approximately 5,000 high quality Gli binding sites, including all known Gli-dependent enhancers. In order to identify functional target genes, we intersected the DNA binding data with gene expression profiles, generating a list of 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing direct targets of both Gli-activator and Gli-repressor based transcriptional control. These studies provided the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb (Vokes et al., 2008).
In our current experiments, we are interested in identifying specific classes of Gli target genes in the limb and determining co-factors that might be involved in their regulation. A second major interest is understanding how multiple signaling pathways in the limb are integrated at the gene level. We are defining binding sites for the transcriptional inputs of additional major pathways active in the developing limb. We believe that the intersection of these datasets will help us understand how this integration occurs and will also highlight the most critical genes in these processes. It is our hope that these studies will help illuminate the processes underlying certain classes of congenital birth defects. |
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