This research program focuses on the role of intercellular communication via peptide growth factors in animal embryonic development. Using the tools of genetics and molecular biology, our goal is to describe biochemical pathways through which growth factors act to modulate morphology, gene expression and cell behavior. The major focus is on the Transforming Growth Factor beta family and the Wnt pathway of secreted signaling molecules. Results are relevant to the use of growth factors as therapeutic agents in and of themselves, and as cytokines in the manufacture of cellular products. In addition, understanding the stability of differentiated cells and their capacity to be reprogrammed by their environment is a major concern in use of cellular therapies, particularly stem cells. Our work uses the fruitfly Drosophila melanogaster as a model system. All growth factors signaling pathways examined to date (TGF-beta, Wnt, FGF, EGF) are highly conserved between Drosophila and mammals. Major projects: a) Use of genetic interaction screens to identify components of the TGF-beta signal transduction pathway. The major TGF-beta family member in Drosophila, the product of the decapentaplegic (dpp) gene, is the TGF-beta family member best characterized by mutational analysis. Mutant phenotypes exist that reflect the many requirements for this growth factor during embryogenesis. I have discovered a class of adult viable mutations whose severity is closely linked to the functional level of dpp signaling. These mutations therefore serve as a barometer of signal transduction pathway activity and have been used to screen the Drosophila genome for loci that interact with dpp in this pathway. One of the screens used an existing bank of mutations created by P element transposon-tagging to facilitate rapid recovery of identified loci. With the completion of the sequence of the Drosophila genome, we hope to be able to correlate interacting loci directly with putative protein coding regions. Some of the interacting mutations are in previously studied genes, such as transcription factors, cell cycle regulators, cellular adhesion molecules, and chromatin remodeling factors. Cell biological and genetic techniques will be employed to understand the nature of these interactions. b) Biology of the head capsule phenotype. The mutations used for the above described genetic screens alter the head capsule of the fly. Various external structures such as the eyes and sensory organs are reduced, eliminated or duplicated. The adult head derives from paired epithelial sheets called imaginal discs that are elaborated during embryogenesis and then undergo final differentiation at the metamorphosis of the pupa. Imaginal discs have become a popular model system for studying signal transduction as they have complex pattern formation, but unlike the embryo, also undergo growth, allowing this aspect of signal transduction to be studied. During the last year we have mapped the lesions in the DNA associated with our mutations and we have sequenced one specific mutation. This mutation appears to alter putative binding sites for several homeodomain transcription factors. One of our goals in the next year is to identify which homeodomain transcription factors interact with these sites to regulate dpp's expression in the head, and what potential feedback exists between transcriptional regulation and signaling molecules. In addition, with the identification of the region of the dpp gene which is altered in head capsule mutations, we have been able to build a Beta-galactosidase reporter construct where expression is driven by cis regulatory DNA from this region. This construct has been used to create transgenic flies. Expression of this construct is limited to the primordia of the adult head, as would be predicted. In addition, the expression pattern is consistent between the structures altered in the mutant phenotype and the established developmental fate maps for the adult head. These data confirm that we have identified the correct region of the dpp gene involved in head development, but also provide a valuable reagent to look at genetic interactions that might alter the expression of the reporter construct in the head primordia of the fly. The sophisticated genetic reagents available in Drosophila allow examination in the whole organism of both classic loss of function mutations and also ectopically expressed genes of interest in almost any tissue. This type of analysis will allow us to screen candidate genes for their ability to effect dpp expression in the target organ: the head. The expression pattern suggests to us that dpp's action in the adult head fates the entire ventral surface of this structure. This is an area of fly development that is very poorly understood. Most of the genetic interactions that have been described for the elaboration of fly tissues, such as the nervous system and limb development, are conserved across species line to vertebrates, thus it is possible that data derived from our analysis of Drosophila head development will shed light on genetic interactions relevant to human head (and brain) development. c) Transcriptional control of a TGF-beta responsive gene. Dpp participates in the formation of the alimentary system of the fly larva, particularly in the morphogenesis of the midgut. Over the past 10 years I have described the factors responsible for dpp's expression in this tissue. The enhancer elements that control transcription are directly controlled by the HOX ortholog Ultrabithorax, a homeodomain-containing transcription factor. In addition, the homeodomain transcription factor extradenticle, homologous to the mammalian oncogene PBX, is required for enhancer function. We have recently shown that the WNT pathway controls transcriptional repression though the HMG box factor Drosophila TCF. Many years ago I showed that dpp expression was autoregulated in this tissue and we have examined sites within the enhancer for the recently described TGF-beta responsive transcription factors, the SMADS. Interestingly, alteration of canonical SMAD binding sites does not affect gene expression, suggesting that dpp's autoregulation is carried out through other factors. We are also interested in understanding how multiple signal transduction pathways converge on a given enhancer element and would like to know if this happens though synergy of transcription factors bound to DNA, or by a more complex mechanism of posttranscriptional modification of transcription factors via cross-talk between the two pathways.
