Kristi A. Wharton, PHD, MS, MPhil - US grants

This proposal is intended to initiate a research program to study cell signalling during Drosophila development. The wealth of genetic information and the ease of molecular manipulation in Drosophila make it an ideal system for the dissection of processes involving many components, such as signal transduction pathways. Here Dr. Wharton proposes to elucidate the function of the newly identified TGF-beta like gene, 60A, through genetical and developmental analyses. Mutations in the gene will be generated in an F2 lethal screen and a male sterile screen. The developmental consequences of these mutants will be determined by direct observation of their development, cuticular preparations, histology and in situ hybridizations. Potential genetic interactions involving 60A, and other genes which code for putative components of a TGF-beta signal transduction pathway, such as, a second Drosophila TGF-beta like gene, dpp, will be examined. The results of these experiments will allow us to better understand the roles of TGF-beta like proteins in communication between cells during development.

DESCRIPTION (provided by applicant): BMPs, the largest family of secreted signaling molecules within the TGF-beta super-family, regulate many processes critical for organismal development, as well as tissue repair. Many components of the TGF-a and BMP signaling pathways are directly implicated in a number of human diseases, most notably heritable neoplastic vascular disorders and gastrointestinal neoplasias. As morphogens, many BMPs are capable of eliciting diverse cellular responses, thought to be determined by the level of signaling. Interestingly, several human disorders and syndromes have recently been attributed to abnormal levels of signaling by BMPs or TGF-betas. In studies of development, the morphogen gradient theory has been useful in guiding research aimed at understanding how positional information and the specification of multiple cell fates are achieved. Despite our extensive knowledge of which molecules are important in patterning, such as BMPs, our understanding of how gradients of morphogenetic information are generated and interpreted is surprisingly limited. Data from our laboratory shows that the BMP activity gradient responsible for patterning the Drosophila wing is generated by the differential contribution of two BMPs, Gbb and Dpp. Dpp exhibits short range signaling activity to pattern cell fates near its source in the central domain of the wing imaginal disc, while Gbb exhibits long range signaling activity to pattern elements found far from its source. The experiments outlined in this proposal are aimed at identifying the molecular basis of this difference in range of signaling. Our aims are: Aim 1: Identify the protein domains of BMP ligands important in determining functional range; Aim 2: Identify sequences and processes critical for regulating functional range; and Aim 3: Elucidate the relationship between physical range and functional range. The unique strength of the proposed structure/function analysis is that function will be assayed in vivo, at endogenous levels, in the normal context of the developing organ. Such an analysis is essential for furthering our understanding of BMP signaling, given the extreme sensitivity of cells to the level of signaling output from this pathway. The role of the BMP type I receptors in generating the BMP activity gradient will be determined, as will the effect of BMP ligand co-expression. Results from these experiments will have a significant impact on our understanding of how BMP signaling levels are regulated.

Project Summary/Abstract The morphogen gradient theory has been a useful framework in guiding studies aimed at understanding cell identity is generated across a developing tissue. Studies investigating the role of BMP signaling in the specification of different cell fates across the developing Drosophila wing imaginal disc have proven that this system is an ideal one to study the molecular mechanisms governing morphogen gradient establishment, maintenance and interpretation. Our previous research uncovered three important features of this gradient system, first, we found that two morphogens, Dpp and Gbb, contribute in distinctly different ways to the BMP activity gradient;second, Sax, one of two receptors mediating morphogen signals has a dual function of both blocking and promoting signaling; and third, a transcriptional feedback mechanism buffers the signaling gradient against stochastic or environmental fluctuations. In this renewal application, we propose a research program to extend our findings with regard to the different behaviors of the BMP signaling molecules, Gbb and Dpp and the novel behavior of the Sax receptor. BMPs have profound functions in development and homeostasis, from early embryonic axis specification to the induction of bone growth. Mutations in various components of the BMP signaling pathway are responsible for multiple diseases and syndromes, including juvenile polyposis, brachydactyly, FOP, HHT2, Loeys-Dietz syndrome and pancreatic carcinomas. Clearly, misregulation of the BMP signaling pathway has serious ramifications on human health and development, and given their potent effects on cell physiology, BMP ligands have long been a desired therapeutic agent. However, to ensure success as therapeutics or in disease intervention, the action of these powerful molecules must be understood in the context of the whole organism. The BMP pathway is highly conserved throughout the animal kingdom, at both the molecular and functional level. This allows us to make use of the Drosophila model system to more quickly investigate not only the factors but also the mechanisms responsible for regulating BMP signaling activity. We have the advantage of examining the interplay between different signaling components at their endogenous concentrations, in their normal location, not possible in most other experimental systems. We have been able to identify new components and novel biochemical behaviors that impact BMP function and intend to investigate the mechanistic underpinnings of these new findings. Our results can easily be extrapolated into the human system and will provide valuable insight into not only our understanding of BMP signaling but also into the intricacies of morphogen gradients as a fundamental mechanism by which different cells acquire their identity.