This research proposal addresses the problem of genetic control of development in mammals using the laboratory mouse as a model system. It will take advantage of recent progress made in the analysis of developmental control genes form Drosophila, which lead to the isolation of a cross homologous, protein coding DNA sequence termed as homeo box. The fact that this highly conserved sequence has so far been found exclusively in Drosophila genes with developmental control functions, and the demonstration of its widespread occurrence in the genomes of other animal groups including higher vertebrates, lead to the speculation that the homeobox sequence may serve as a molecular probe for the identification of developmental control genes also in mammals. Provided that this assumption is correct, the murine homeo box containing gene family may serve as a model system for studying genetic control mechanisms of mammalian development. The long term objective of the proposed research is to characterize structure and function of a previously isolated member of this gene family, designated as Hox 3.1. Since homeobox genes are believed to control the expression of target genes via trans regulatory mechanisms, one set of experiments will focus on the isolation of Hox 3.1 specific target sequences by protein/DNA binding studies, which will employ Hox 3.1 protein produced in a gene expression vector system. In addition, antibodies raised against this protein will be used for localizing Hox 3.1 protein in tissue sections. A second approach for the isolation of Hox 3.1 specific target genes will be based on subtraction hybridization of cDNAs derived from Hox 3.1 expressing and nonexpressing culture cells, carrying an inducible Hox 3.1 gene construct. More direct evidence for a developmental control function of Hox 3.1 may come from experiments using site directed mutagenesis in pluripotent embryonic stem cells with the goal to generate chimeric mice by blastocyst injection. These mice may transmit the mutated Hox 3.1 gene through their germ line, leading to heterozygous offspring that may then be used for further analysis of possible developmental defects by proper breeding. A second set of germ cell transformation experiments aims at the identification of cis regulatory elements mediating region specific expression of Hox 3.1. A series of DNA constructs containing progressively deleted segments of the putative Hox 3.1 5' regulatory region linked to a reporter gene will be used for the generation of transgenic mice or mouse embryos. Differences in the expression patterns of the reporter gene may then be correlated with the presence of defined 5' DNA segments.
