The SHH morphogenetic pathway exerts multiple functions during embryonic and adult life. It is involved in various cellular contexts to provide positional information, but also controls cell survival, proliferation and differentiation. Aberrant activation of the pathway has also been linked to tumour formation and growth. The SHH signal is integrated by receiving cells via the GLI transcription factors, which regulate the expression of SHH target genes. In particular, the GLI3 protein is processed into a repressor (GLI3R) form in the absence of SHH, while is converted into a transcriptional activator (GLI3A) upon exposure to SHH. SHH being a secreted factor, it is thought to establish a dual GLI3R:GLI3A gradient, with high levels of the GLI3R away from the source and high GLI3A close to the SHH-expressing cells. This mechanism permits various levels of activation of the pathway, depending on the GLI3R:GLI3A ratio. Despite the initial description of many pathway target genes, the primary response to variable SHH doses over time, and the subsequent alterations in GLI3R:GLI3A isoform ratios, are poorly understood. We have manipulated the Gli3 locus in mouse ES cells by RMCE to introduce a FLAG-tag into the endogenous protein. We have evidence that this FLAG-tagged GLI3 creates a sensor of the SHH signal (GLI3A-FLAG). We will use neuralized embryoid bodies and the knock-in mouse line derived from GLI3A-FLAG ES cells to monitor the cellular and molecular dynamics of GLI3A in sensing SHH activity. This system will permit us to track the relative occupancy of the promoter/regulatory elements of SHH target genes by GLI3, allowing a real-time readout of the way cells respond to SHH over time. Moreover, we will be able to visualize and study the postulated Gli3A gradient in vivo. Funding from the ERG program, together with the long-term contract I have been offered, would be a significant asset in establishing myself as an independent reseacher.
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