Genetic Control of Cerebellar Zonal Patterning

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

Much is known about the molecular and genetic mechanisms controlling the regionalization of the developing brain, but much less is known about how this information ultimately gives rise in maturity to unique brain regions with distinct functions. One strategy to approach this is to dissect the molecular and genetic mechanisms controlling the expression of region-or cell-specific genes whose products contribute directly to a unique functional property of cells in the mature brain. The rodent cerebellum is ideal for the understanding of these mechanisms as there is a rich historical record of natural mutations affecting the development and function of this brain region. In addition much is known about the precise neurobiological functions of its constituent. The purpose of our proposed experiments will be to determine the molecular mechanisms of expression of the L7/Pep-2 gene in the rodent cerebellum. In the mature brain this gene is expressed only in cerebellar Purkinje cells and not in any non-brain tissues. In addition, the gene is expressed in a transient pattern of stripes in the cerebellum during the development and this pattern reflects the well know-known zonal wiring diagram of the cerebellum. Furthermore, the L7-Pcp-2 protein carries a GoLoco-domain and functions as a modulator of GDP binding to large heterotrimeric G-proteins suggesting a role of the protein in modulation of Ca2+channels, a major component of information flow in Purkinje cells. We have combined a novel in vivo expression strategy using transgenic mice with comparative genomics to identify key DNA sequence elements within the promoter (expression control region) of the L7/Pcp-2 gene. The data show very high conservation across species of both the sequence and spacing of seven elements within a short region of this promoter. One of these elements is a binding site for the orphan nuclear receptor, RORa, a likely modulator of Ca2+ metabolism genes in bone that is also known to be expressed in Purkinje cells. The aims will be i) to determine more precisely the role of these elements by expression testing in further transgenic mouse studies, ii) to use FPLC purification and MALDI-MS protein sequencing to identify the key factors that bind to these elements, and III) to test the effect of null mutation of these factors on the expression of L7/Pcp-2. These studies provide the best opportunity to-date to identify a precise molecular complex controlling both cell-specific and G-protein pathway-specific gene expression in the brain.