Our goal is to learn the molecular mechanisms by which genes control the development of multicellular structures. Homeotic genes are among the most important regulators of pattern formation, mutations in them causing dramatic transformations of one part of an animal into the likeness of another part. Different alleles of the Drosophila Antennapedia (Antp) homeotic gene, for example, transform antennae into legs, legs into antennae, or eyes into wings. The homeotic genes are master regulators, coordinating the transcription of other genes to control morphogenesis during embryonic development and metamorphosis. The precise spatial and temporal patterns of transcription of homeotic genes are crucial for directing cells into different developmental pathways. The patterns are initiated under the control of segmentation genes and are then maintained and modified by interactions among the homeotic genes and by Polycomb group and other genes. We are using promoter-lacZ gene fusions, introduced into flies, to investigate the regulation of the two Antp promoters. We will search specifically for cis-acting Antp sequences regulated by known segmentation genes and for sequences involved in maintaining precise patterns of expression. The protein products of homeotic genes are transcription factors but little is known about the """"""""target"""""""" genes regulated by the homeotic proteins. We have found that two growth factor-like proteins, encoded by the dpp and wg genes, are regulated by homeotic genes. To obtain additional information about target genes we will take advantage of a powerful new tool, the localization of homeotic proteins on polytene chromosomes in vivo. Homeotic proteins with very similar DNA binding domains direct the formation of very different segments of the fly. Our in vivo assays reveal the different homeotic proteins distributed in distinct patterns on the polytene chromosomes, demonstrating a high degree of specificity and suggesting that the proteins control different arrays of target genes. One putative target gene identified in this way is in fact regulated by the homeotic protein which binds there. By transposing fragments of the target gene DNA to new chromosomal sites the sequences to which the homeotic protein binds can be mapped. The polytene assay as well as biochemical methods will be used to answer four questions: What DNA sequences are regulated by the different homeotic proteins? Are known gene interactions, such as cross-regulation among the homeotic genes and the regulation of the growth factors dpp and wg, due to direct protein-DNA interactions? What aspects of the proteins' structures are essential for determining the specificity of the proteins' interactions with their targets? What are some of the target genes and how do they contribute to morphogenesis? These studies will lead to a better understanding of the transcriptional control of morphogenesis.
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