The mouse mutant """"""""rostral cerebellar malformation"""""""" (rcm) is ataxic and has within the cerebellum a reduction of the size and number of folia as well as localized disruptions of laminar structure. The applicant has obtained a transgenic line in which an insertion in or near rcm provides a basis by which the gene can be cloned. The objective of this grant is to identify a cDNA encoded by the rcm gene, study the distribution of rcm in the embryonic and early postnatal cerebellum, assay the effects of the rcm mutation on other genes known to affect cerebellar development (and visa versa), and to determine whether rcm might play a role in permitting cerebellar granule cell migration. The work described in the original proposal is divided into two specific aims:
Specific aim 1 :
The first aim was to confirm that the candidate gene is rcm. The results described in the supplement make this aim moot, it has already been accomplished. The similarity in rcm to unc-5 is very interesting. It is further proposed to determine the temporal and spatial expression pattern of rcm mRNA in the developing cerebellar cortex by in situ analysis of P0-P15 cerebella. An attempt will be made to raise polyclonal antibodies against bacterially expressed rcm protein. These antibodies would be used to visualize the protein on cerebellar sections. The antibodies would also be used on an organ culture assay of cerebellar cell migration to determine if they prevented the granule cells from migrating from the external to the internal granule cell layers.
Specific aim 2 : The effects of various genetic backgrounds will be evaluated for their effects on the severity of the rcm phenotype. In addition, mutant embryos will be analyzed to determine the nature and timing of the earliest observable defect in neural development. In addition, in situs for rcm mRNA will be performed in embryonic preparations as above. Finally, the expression pattern of rcm will be examined in other mutant lines in which cerebellar development is perturbed: e.g. en1, en2, Wnt1, reeler, and weaver. Conversely, in situs directed against these gene transcripts will be analyzed in the rcm mutant. The hope is that coincident changes in expression patterns of pairs of genes will suggest that they act within the same developmental pathway.
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