The ability of organisms to control signaling between cells is critical to the formation of precisely defined cell types, and to thereby control the size and patterns critical to the formation of tissues and organs during both embryonic development and adult life. Failures in the regulation of that signaling are known to underlie human birth defects and a variety of important health problems, including the excessive cell growth that causes cancers. One of the most important of the signals between cells is carried by a family of molecules termed Bone Morphogenetic Proteins (BMPs); in fact, the BMP signals are critical to the normal development of almost all animal species. However, the way in which BMP signaling is regulated is still poorly understood. This project involves a series of studies that will identify and characterize previously unknown regulators of BMP signaling. It uses for these studies the fruit fly, Drosophila melanogaster, since the genetic and molecular manipulations possible in this organism are unmatched by any other experimental system. The studies make use of a Drosophila tissue exquisitely sensitive to the loss of BMP signaling; the loss of this tissue in mutant flies provides a powerful method of identifying defects in novel members of the BMP signaling pathway. These studies have already led to the isolation and characterization of three novel proteins, now known to be shared by humans, that bind to and regulate BMP signals during their passage from cell to cell.
The Broader Impacts of this project are several. It will used to advance discovery by training both graduate and undergraduate students; undergraduates are involved not only in senior-level independent study classes but also as part of the University of Winsconsin's introductory biology course. Many of these students have gone on to productive careers in the biosciences, as graduate students, postdocs or faculty. The project also aids in the promotion of underrepresented groups, and the majority of both graduate and undergraduates so trained have been women. The PI is also heavily involved in introductory and advanced undergraduate education, and presents the research findings to students as part of their introduction to scientific methodology. The molecular reagents and Drosophila strains developed in the lab are made available to other researchers either directly or via the national stock centers, and the award is used to support the PI's websites on techniques and genetic stocks. The PI has also has used this work to broaden interactions between the biological and mathematical communities through efforts to mathematically model the action of specific molecules.
Signaling between cells is critical for many biological processes, including the specification and modification of cell identities, the control of tissue growth, and responses to infection. Failures in normal cell signaling can lead to birth defects, cancers, and disease pathologies. Despite much progress, however, there are many details the we do not understand about how signaling is controlled- why do cells produce the correct amount of signal, how is that signal transmitted to nearby and distant cells, how do cells respond to those signals, and how do different signals interact? This project examines these questions for a specific family of signaling molecules, the Bone Morphogenetic Proteins (BMPs). Our project identified and characterized previously unsuspected modulators of BMP signaling, using the many genetic and molecular advantages provided by the fruitfly, Drosophila melanogaster. The advantages of using this "model" animal are several: the genetic and molecular analyses possible in Drosophila are perhaps unmatched by any other animal, the analyses can be carried out much more quickly and cheaply than in mammals, and the mechanisms underlying BMP signaling in Drosophila wing shows striking parallels with those used in humans. There is an additional advantage of the Drosophila model: loss of BMP signaling during fruitfly development from embryo to adult often leads to an easily detected defect: the loss of the crossveins from the wing. We showed that crossvein cells require localized BMP signaling within the developing wing, and have used this to identify mutants that are defective in BMP signaling, and then identify the proteins that are defective within these mutants. For instance, we identified for the first time the BMP-binding protein Cv-2/BMPER, and showed it can increase BMP signaling. Mutations in the human gene encoding Cv-2/BMPER are now known to cause a fatal human genetic disorder characterized by severe defects in fetal bone development. The first aim of our project examined how extracellular BMP-binding proteins promote signaling. Our past work identified two distinct systems of BMP-binding proteins: a complex of Sog/Chordin and Cv-Tsg2 that transports BMPs, and a second using Cv-2/BMPER that acts locally to increase binding between BMPs and their receptors. The project funded experiments examining how the binding between the Sog/Chording and Cv-2/BMPER proteins mediates a connection between these two systems. In the second aim we examined a completely new component of the system that we discovered, the Cv-d lipoprotein. The project funded our experiments showing that extracellular Cv-d binds BMPs to another pair of proteins required for the movement of BMPs from cell to cell- the two Drosophila glypicans. Cv-d is quite similar to the human Vitellogenin proteins, and our work provides the first example linking this important family of proteins to the promotion of BMP signaling. Our third aim exploited our ongoing genetic screen to uncover additional regulators of BMP signaling. We discovered a signaling receptor that is required for normal BMP signaling through the creation of the intracellular signaling molecule cGMP. A number of studies from mammals suggest that cGMP can promote BMP signaling, and that this interaction underlies the crosstalk between BMP signaling and the two cGMP mediated by natriuretic peptide and nitric oxide during pulmonary pathologies. However, the mechanism that mediates this crosstalk is still the subject of study. Our NSF project supported our experiments showing that cGMP affects the structure of proteins in the extracellular matrix that lie between wing cells, and which are known to bind BMPs and BMP-binding proteins. Moreover, we can rescue the BMP signaling defects caused by reduction of cGMP by supplementing the components of the extracellular matrix, demonstrating a causal role. This is the first time a connection has been made between cGMP and the extracellular regulation of BMP signaling, and thus we have established a powerful model for future studies on this pathway. The work funded has had an impact on the study of developmental biology in mammals and continues to be relevant for ongoing medical studies of BMP function in human disease and birth defects. The project also has had a wider impact, as it has also been used train both graduate students, postdocs and undergraduates. Undergraduates are involved not only in senior-level independent study classes but also as part of the introductory biology sequence for majors. Many of our trainees have gone on to productive careers in the biosciences, as graduate students, postdocs or faculty. The majority of both graduate and undergraduates so trained have been women. Molecular reagents and Drosophila strains developed in the lab are made available to other researchers either directly or via the national stock centers. The PI maintains a web site detailing techniques for immunohistological techniques in Drosophila as a guide for those new to the field (http://mywebspace.wisc. edu/ssblair/web/antiweb.html). The PI has written many technical guides for the field and reviews on general topics in developmental biology.
