MOLECULAR GENETICS OF SEGMENT IDENTITY

The morphogenesis of a complex organism depends upon the execution of an intricate genetic hierarchy that provides individual cells with positional and cell fate information. In Drosophila the homeotic selector genes play a central role in this hierarchy because they interpret positional information and determine cell fate. These genes have highly conserved homologs in many animals, including humans, suggesting that their analysis in Drosophila may be a key to understanding vertebrate development as well. The molecular dissection of vertebrate development will, in turn, provide insights into human diseases, including birth defects and various cancers, that appear to result from mistakes in the execution of this genetic hierarchy. Drosophila is an especially well suited organism in which to study this hierarchy because of its experimental versatility, particularly with regard to its molecular biology and genetics. The homeotic selector genes provide an attractive entry into these studies because of the large amount of genetic and molecular information already available. The products of these genes all contain the DNA-binding homeodomain and act, at least in part, by regulating the transcription of downstream target genes. The experiments proposed here have two complementary goals: to identify and characterize target genes that are regulated by the homeotic selector gene, Ultrabithorax, and to understand in detail how homeotic selector proteins regulate target gene expression. The first goal will be approached in two ways. A subtractive hybridization approach has succeeded in isolating 20 genes that are induced by Ultrabithorax gene products. Three of these genes appear to be bona fide downstream target genes and will be studied in detail. A genetic approach involves isolating dominant mutations that enhance or suppress Ultrabithorax-induced homeotic transformations. Genes identified in this screen will be examined to determine if they are indeed Ultrabithorax target genes. If they appear to be true target genes they will be further characterized by DNA sequencing, determining their in vivo spatial and temporal expression patterns, and examining their genetic interactions with other homeotic mutations. The second goal of this proposal is to better understand how homeotic selector proteins regulate target gene expression. Ibis will be accomplished by making mutant Ultrabithorax proteins and studying them using in vivo assays that measure homeotic protein functions. Assays are already available for studying functions required during embryonic development. In addition to these, new in vivo assays will be developed to measure Ultrabithorax functions required for adult morphogenesis. These in vivo studies will be paralleled by experiments utilizing new assays that measure the in vitro DNA binding properties of mutant Ultrabithorax proteins. These experiments will determine if particular mutations affect Ultrabithorax functions by affecting their ability to bind DNA or by affecting some other property, such as specific protein-protein interactions.