Mirotsou, Maria; (2001) Functional analysis of the Id4 protein by conditional overexpression. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Id4 protein is a member of a subclass of helix-loop-helix (HLH) proteins, which are involved in the regulation of transcription as well as cell growth and differentiation. It has been shown that during mouse development the Id4 is exclusively expressed to the developing nervous system. In adult tissues, Id4 expression is more widespread and most abundant in brain, kidney and testis. The Cre/loxP recombination system is a powerful tool that allows in vivo modifications of genes in a tissue/time restricted way. This thesis will describe the employment of the Cre/loxP system, in order to assess the importance of the Id4 protein in the development and cell lineage specification of the central nervous system in vivo. In addition, the same system was used to address the question of functional redundancy of the Id genes by ectopically expressing Id4 in thymocytes in vivo First, mice carrying a dormant Id4 transgene were characterised. In these mice, expression of the Id4 transgene is blocked by transcriptional and translation stop signals (TSS), situated between the promoter and the Id4 gene. The TSS are flanked by loxP sites and can be deleted through Cre/loxP mediated recombination. To activate the dormant Id4 gene specifically in neuronal cells, mice were generated by Dr Elisa Cinato. These mice express the Cre recombinase in the developing nervous system and adult brain. Mating of the two transgenic lines resulted in mice, which overexpressed the Id4 protein specifically in the nervous system. In these mice, accumulation of neurons and reduction in glial cells in the cerebral cortex was observed. The shift in the neural cells numbers was accompanied by proliferation and apoptosis. These results establish the importance of Id4 protein in neural cell fate determination and differentiation. Simultaneously, the Id4 transgenic mice were mated with mice expressing the Cre recombinase in thymocyte specific way. The mice generated from these matings overexpressed Id4 in thymocytes. This expression resulted in disrupted thymocyte development and resembled the effect of overexpression of other Id proteins in thymocytes. Since the Id4 is not normally expressed in thymocyte, our results support the idea of functional redundancy of the Id genes.

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