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Setting-up a patterning system: the regulation and temporal changes in functioning of the transcriptional regulator GLI3 during mouse limb bud development

Final Activity Report Summary - GLI3 LIMB PATTERNING (Setting-up a patterning system: the regulation and temporal changes in functioning of the transcriptional regulator gli3 ...)

Sonic hedgehog (SHH) is a secreted protein used by embryonic and adult cells in tissues and organs to communicate by transmitting information concerning e.g. position and identity and to regulate their proliferation and differentiation. Its functions have been best studied in the context of the central nervous system (CNS) and limb organogenesis, where SHH is essential for pattern formation and proliferation. In the limb bud, SHH is involved in the establishment of anterior-posterior axis, which controls the correct development of ulna and radius and the formation of five digits (fingers and toes). Alterations of the SHH pathway cause severe congenital malformations including cyclopia and loss of hands and feet. Furthermore, aberrant, postnatal activation of the SHH pathway underlies a large variety of tumours in humans possibly due to inappropriate SHH-mediated stimulation of cell proliferation and/or altered differentiation states. To ensure that cells do not activate SHH signal transduction and expression of targets in the absence of the SHH ligand, there is cellular gatekeepers that block activation of signal transduction without a cell being exposed to the SHH ligand. The GLI3 transcriptional regulator is such a gatekeeper that negatively regulates SHH signal transduction by repressing the activation of target genes. Mice lacking this Gli3 "brake" die perinatally due to a variety of congenital malformations including digit polydactyly and severe CNS patterning defects.

As this lethality has precluded the study of the functions of Gli3 during per- and postnatal development and adult life in more detail, we have generated a mouse model that allows to inactivate Gli3 in a temporally and/or spatially controlled manner either during embryogenesis or after birth. Using this so-called conditional Gli3 allele, we are now dissecting the functions and temporal requirement of this SHH gatekeeper during limb and brain development in mouse embryos and in SHH-dependent tumorigenesis after birth.

Using a novel technique (RMCE), we have also generated additional alterations in the Gli3 locus, that allow us to detect and purify the GLI3 containing transcriptional complexes from intact embryonic and adult tissues. This systems biology-type approach in combination with cell-biochemistry aims to analyse of the functions and interactions of GLI3 during embryogenesis, tumourigenesis and in adult tissues and stem cells.