ROLE OF POU-FACTORS IN NEURONAL DEVELOPMENT

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The primary objectives of the proposed project are (i) determination of the role of POU factors such as the Drosophila factor Cf1a in development, (ii) characterization of how the Cf1a gene is regulated and its relationship to other pattern formation genes and (iii) confirmation of the putative relationship between Cf1a and the neuron-specific expression of the dopa decarboxylase (Ddc)gene. The proposed specific aims are to characterize the Cf1a transcription unit by mapping and sequencing of CF1a genomic clones and construction of P-element transformant strains expressing Cf1a/beta-galactosidase fusion genes. Analysis of Cf1a/beta-galactosidase fusion genes with mutations within Cf1a regulatory sequences should identify sequences necessary for wild-type expression. The wild-type expression pattern of the Cf1a gene during development will be characterized both by the development of a Cf1a-antiserum for immunohistological labeling, the expression of Cf1a/-galactosidase fusion genes in P-element transformant strains and by continued application of in situ hybridization using digoxigenin-labeled Cf1a probes (see Preliminary Results). The phenotype of mutations within the Cf1a gene will be analyzed by the generation of Cf1a P-element insertion mutants using hybrid dysgenesis as well as a thorough genetic characterization of overlapping deficiencies which move the Cf1a gene. The phenotypic effects of altering the restricted expression of Cf1a by ubiquitously expressing the Cf1a protein under the control of the hsp70 promoter in heat-shocked P-element transformant strains will determine whether restricted expression both spatially and temporally is required for the correct function of Cf1a. A conclusive demonstration that Cf1a directly regulates Ddc expression in specific neurons would be especially significant since it would like a lineage determinant to regulation of a downstream gene involved in the phenotypic expression of a neuronal cell-type. The ubiquitous expression of Cf1a protein could also potentially demonstrate a direct effect upon Ddc gene expression of abherrant CF1a expression causes Ddc expression in inappropriate neurons. If this effect is dependent upon an interaction with the Cf1 binding site, then it would not be seen in transformant strains containing only a Ddc gene with a clustered point mutation in the Cf1 binding site, rendering it incapable of binding the Cf1a protein. A correlation of results from studies on how Cf1a is regulated with the phenotypic effects of over- and under-expression of the Cf1a gene in mutant strains will suggest possible developmental functions for POU factors such as Cf1a. The remarkable conservation of structure between human and Drosophila POU-domains suggests that results from molecular studies in Drosophila can be readily extrapolated to more clinically relevant applications. The suspected role of the Cf1a gene in the development of dopaminergic neurons should be of interest for clinicians investigating various human pathologies such as Parkinsonism which involve the abherrant development of premature degeneration of specific neurons.