The clustered protocadherins (Pcdhs) are members of the cadherin superfamily of cell surface adhesion molecules and are proposed to play a critical role in the development of the nervous system.
Previous work using whole brain samples has shown that expression levels of the clustered Pcdhs are disrupted in Smcdh1 mutant mice. Since the clustered Pcdhs are subject to random combinatorial monoallelic expression in individual neurons so that each expresses a unique combination of Pcdh isoforms, we will determine whether the combination of Pcdhs expressed is disrupted at the single cell level in mutant mice. To do this, we have been generating mice with Z/EG and Pcp2-Cre transgenes that will label the Purkinje neurons with green fluorescence protein (GFP); and we will isolate individual GFP labeled Purkinje cells for single cell qRT-PCR analysis.
Using cultured neurons would make it much easier to isolate individual cells for analysis. It would also allow us to genetically manipulate the cells much easier than using animal models. Cultured mouse neuronal stem cells (NSCs) and NSCs differentiated into mature neurons will be used to study the epigenetic mechanism underlying the random combinatorial monoallelic expression of the clustered Pcdhs that is disrupted in Smchd1 mutants. We have conducted qRT-PCR for all of the clustered Pcdhs to prove their expression is disrupted in Smchd1 mutant NSCs. This study has shown that the expression of some clustered Pcdh genes (Pcdh-α and part of Pcdh-β) are disrupted in Smchd1 mutants even in undifferentiated NSCs.