The objective of this program is to identify and functionally characterize neurogenic genes that are required for CNS development. Given the high conservation in basic developmental mechanisms used by all metazoans, we have focused our efforts on the study of CNS development in the fruit fly (Drosophila melanogaster), where the genetic information required for these events is accessible. Using classical genetic, molecular biology and transgenic techniques, we have continued to study the function of genes expressed during neuroblast lineage differentiation. Thus far, our study of castor (cas), a novel Zinc finger gene required for proper CNS neuroblast development, has revealed that it encodes a nuclear located, sequence-specific DNA- binding protein whose expression is restricted to late forming CNS neuronal precursor sublineages. We hypothesize that the Cas protein functions as a transcription factor required for correct regulation of genes in these cells. Our recent studies have shown that cas controls cell fate decisions by regulating the expression of all known POU genes during CNS development. POU transcription factors establish neuronal identities in metazoans, yet little is known about the regulatory networks controlling their expression. While Cas is required for the silencing of both pdm-1 and -2 gene expression (early lineage determinants), the drifter and I-POU genes require Cas for their full expression. Cas shares DNA-binding specificity with another pdm repressor, the gap-segmentation gene regulator Hunchback (Hb). Our studies reveal for the first time that all CNS ganglia contain sequentially layered neuroblast progeny subpopulations that can be distinguished by their expression of either Hb, Pdm-1 or Cas. Hb and Cas may directly silence pdm expression in early and late developing neuroblast sublineages, respectively, as pdm-1 regulatory DNA contains about 32 Hb/Cas binding sites and its enhancer(s) are ectopically activated in cas- neuroblasts. By ensuring correct POU gene expression boundaries, hb and cas maintain temporal subdivisions in the cell- identity circuitry controlling CNS cellular diversity. An enhancer-trap screen for additional genes required by late neuroblast sublineages has yielded a transformant line that expresses beta-gal in a subset of these cells. We have mapped its P-element integration site to the second chromosome at map position 45F; set up P-element mobilization screens to isolate imprecise-excision mutations; cloned 25 kb of genomic DNA surrounding the P-element insertion; and, are now screening cDNA libraries. This enhancer-trap line has a homozygous viable neurologic defect, presumably due either to a hypomorphic or knockout mutation caused by the P-element insertion. When compared to wild type behavior, both larvae and adults are less active. Mutant adults do not fly and, although they exhibit other normal behaviors, their locomotion during these activities is significantly reduced. Additional tests are underway to characterize this phenotype.
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