STERIOD HORMONE-REGULATED GENES IN THE CNS

The proposed experiments are part of a long-term plan to investigate the
genetic control of nervous system remodeling during development. Because
of the combined power of molecular and classical genetic approaches to
developmental problems, Drosophila CNS metamorphosis has been chosen as an
experimental model system. Steroid hormones influence many aspects of CNS
development in vertebrates and invertebrates. Determining the mechanisms
mediating hormonal effects on CNS morphogenesis, as well as on neuronal
birth, differentiation, and death, are critical to understanding
abnormalities of human nervous system development. In keeping with the known action of steroid hormones on gene expression,
mutants of a gene regulated by the steroid molting hormone 20-
hydroxyecdysone (20HE) exhibit specific defects in regional CNS
morphogenesis and neural organization. this gene, the Broad Complex (BR-
C), is expressed in many tissues, including the CNS, and regulates the
activities of many other loci. The proposed experiments are based on the
hypothesis that CNS defects in the mutants result from the misregulation of
BR-C target genes in the CNS. Thus, the major goal of this project is the
isolation and characterization of 20HE-inducible genes expressed in the
CNS, with particular emphasis on those regulated by the BR-C. Initially, BR-C transcription in the CNS will be studied by in situ
hybridization and confocal microscopy. The structure and hormone-
inducibility of these transcripts will be analyzed by standard RNA blotting
and mapping methods. The scheme for isolating hormone-inducible genes from
CNS RNA combines the time-honored approach of differential hybridization
with newer cDNA cloning vectors which allow in vitro synthesis of cRNA.
This amplified cRNA provides high-sensitivity reagents for library
construction and analysis. A subtracted cDNA library, enriched for
sequences preferentially expressed in hormone-exposed CNS tissue, will be
constructed and screened. A variety of criteria, including misregulation
in BR-C mutants, will be used to determine which 20HE-inducible clones
represent genes with probable important roles during CNS metamorphosis.
The expression patterns and DNA sequence of these genes will be determined
and their function assessed by the generation and analysis of mutants.