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Interpreting the SHH signal: real-time monitoring of the dynamic regulation of GLI3

Final Report Summary - SHH PATHWAY SENSOR (Interpreting the SHH signal: real-time monitoring of the dynamic regulation of GLI3)

The Hedgehog signalling pathway plays a critical role during the development of the vertebrate embryo. Amongst other structures, the SHH secreted protein is involved in formation of the limb skeleton, and loss of SHH activity leads to severe limb truncations both in human and mouse. Depending if a cell is exposed or not to the SHH signal, the profile of expressed genes changes, and such modulation of the cellular response to SHH depends on the GLI transcriptional regulators. Therefore, we set out to characterise those genes that are directly regulated by GLI proteins during limb organogenesis. In particular, we were interested in the role of GLI3 during limb bud development, as its deficiency results in aberrant activation of Hedgehog signal transduction and polydactyly (appearance of extra digits), both in mouse and humans. To do so, I developed a series of mouse genetic tools that would allow us to:

1) detect the endogenous GLI3 protein isoforms with high sensitivity;
2) inactivate the Gli3 gene in a spatio-temporal controlled manner.

By using a combination of genetic, molecular, and cellular assays, we discovered that GLI3 curbs the proliferation of limb bud mesenchymal progenitors by directly regulating the cell cycle. In particular, the endogenous GLI3 proteins interact with the cis-regulatory region controlling Cdk6 expression. In addition, GLI3 also directly regulates Grem1 expression, a secreted antagonist of BMP activity, which regulates the formation of the cartilage elements prefiguring the limb skeleton. Our genetic analysis revealed that GLI3 is an essential gatekeeper that controls both the proliferative expansion of progenitors and assures their timely differentiation into chondrocytes. In other words, in the absence of Gli3, the progenitors giving rise to the digits (= fingers) do not know when to exit proliferation and start to form the cartilage elements. This explains why inactivation of Gli3 results in production of too many cells and ultimately the formation of extra fingers and toes. Therefore, Gli3 is a critical component in the molecular networks that restrict digit number to five. As mutations affecting the human GLI3 gene also result in to polydactyly (such as in the Greig's cephalopolysyndactyly and Pallister-Hall syndromes), it is likely that alterations of the cell cycle and/or the onset of cartilage differentiation underlie the variable polydactylies observed in human congenital limb malformations.

Our research has been focused on the functions of GLI3/SHH target genes during embryonic limb development, but our study is likely of relevance for other biological systems depending on the Hedgehog pathway, such as maintenance of adult stem cells and malignant progression of certain types of tumours such as medulloblastoma (one of the deadliest childhood cancers) or basal cell carcinomas. For instance, some of the direct GLI3 target genes we know to regulate cell proliferation are potential therapeutic targets for interfering with cancer cell proliferation (such as e.g. Cdk6). Furthermore, the epitope tag we have inserted into the endogenous GLI3 protein has enabled us to identify direct targets of SHH signalling and the relevant cis-regulatory regions in both wild-type and different mutant contexts using both candidate genes and genome-wide approaches. This is an important tool for gaining insight into how cells integrate SHH signalling inputs into a transcriptional response that e.g. controls the balance of proliferation and differentiation of limb bud mesenchymal progenitor cells.