FUNCTIONAL CHARACTERISATION OF THE ADAPTOR PROTEIN SHCD/RALP

Development requires the strict coordination of signalling pathways driving proliferation, differentiation and migration. Adaptor proteins play an important role in the regulation of specific signalling pathways by linking activated cell-surface receptors to their downstream intracellular targets and mediating crosstalk mechanisms between pathways. ShcD/RaLP, the latest identified member of the Shc family of adaptor proteins showed a unique expression pattern from the other members of the family. It is expressed early during embryogenesis in the pluripotent epiblast and is re-expressed in the developing central and peripheral nervous system and its expression is confined to the brain in the adult. In the pathological context, RaLP is expressed in melanomas and its expression level correlates with disease progression. The physiological role of this protein is still largely uncharacterised. The aim of this PhD project was to characterise the function of RaLP and to understand how this family of adaptor proteins have evolved to modulate distinct signal transduction within different cell types. This could also provide insight on how its deregulation could contribute to pathogenesis. RaLP knockout (KO) and heterozygote (HT) embryonic stem cells (ESCs) were used as the in vitro model system to dissect the role and signalling pathway of RaLP in development. We found that RaLP expression was recapitulated in vitro as in vivo as it was tightly regulated during the formation of the epiblast in several differentiation programs and then re-expressed when neuronal commitment initiated. Using the recent protocol that allows the transition of pluripotent ESCs to another pluripotent stem cell type, epiblast stem cells (EpiSCs), we specifically investigated the role of RaLP in the establishment of this cell identity. We found that the genetic deletion of RaLP resulted in an impairment of the establishment and maintenance of EpiSCs due to defective proliferation capacity upon differentiation. Furthermore, we have also described for the first time, the emergence of Cdx2 expressing cells during ESC commitment to EpiSC fate and we observed that the absence of RaLP results in an enrichment of this population and enhanced levels of MAPK/Erk1/2 activation. Our data suggests that RaLP plays an important role in the switch of key pathway/s involved in determining EpiSC identity and that the absence of RaLP perturbs the commitment process. RaLP is also implicated in the differentiation of neural stem cells to neuronal fate. As RaLP is highly expressed in the brain, we sought to understand whether it plays a role in this commitment process. We found that the RaLP KO NSCs showed severe defects in neuronal formation and we are currently analysing our RaLP KO mouse model to obtain a further understanding of the physiological role of RaLP. In summary, our studies showed that RaLP is involved in EpiSC formation and maintenance as well as neurogenesis, and this dual role is unique for this member of the Shc family, providing an interesting insight into the mechanisms that the cell has evolved to regulate signalling pathways through adaptor proteins in a cell specific manner.