This proposal is designed to explore the networks of genes that are required by the central nervous system to build a cerebellum. Targeted mutation and ectopic expression of a variety of pattern formation genes has given hints to the genetic pathways linking the major players that establish the cerebellar and tectal fields. For example, null alleles of Engrailed-1 and Pax-2 are reported to completely block development in this region. During the past project period we lave discovered that the Engrailed-1 phenotype is completely suppressed by transferring the null allele from a 129/Sv genetic background to C57BL/6. A careful review of the literature reveals a similar (though less dramatic) effect of this same background switch on a Pax2 null allele. This stunning finding means that the genetic network can be altered to abrogate the effects of a major developmental defect. Finding the origins of this rescue will be a major goal of the coming project period. Using regularly spaced MIT markers as mapping tools, we will locate the chromosomal loci involved. Expression of candidate genes from the region will be viewed by whole mount in situ hybridization and compared between C57BL/6 and 129/Sv embryos of identical age. Proof of gene involvement will be achieved by using BACs from C57BL/6 library as transgenes to rescue 129/Sv-Enl-/- embryos. The findings from our Engrailed-1 study will guide a second, more efficient probe of the Pax2 phenomenon. A second, related approach to the identification of the suppressor genes is to create En1 and Pax2 chimeras and analyze the behavior of the mosaic nervous system at embryonic days 9.5, 4.5 and as adults. This experimental approach will reveal those aspects of the network that are responsive and those where are intrinsic and hard-wired. Favorable animals will also provide hints as to the distances over which groups of cells can influence their neighbors. To complement the studies of these early events, we propose to study the genetic regulation involved insetting up the 'compartment-like' modules of the adult cerebellum. We have cloned the Zebrin II gene and identified it as aldolase C. During the previous project period we have have used transgenic mice to demonstrate that a 1.8kb basal promoter construct is sufficient to restrict transgene expression to the CNS. A strong astrocyte enhancer is also found in this region. We have further shown that the 3.2kb immediately 5' to this basal promoter contains a Purkinje cell enhancer but the element that results in the banded aldolase C expression pattern is outside even this longer element. We will subdivide the 3.2kb region to more precisely identify the Purkinje cell enhancer. BAC transgenic mice will be used to identify the cis-element responsible for the banding.
Showing the most recent 10 out of 36 publications
