The goal of this project is to investigate how the highly conserved TGF-beta signaling pathway functions, using the embryo of the leech as a convenient model. The BMP sub-family of TGF-beta signaling is active throughout vertebrate development, ranging from dorsoventral patterning and organogenesis to the induction of teeth and hair follicles. Many developmental defects, cancers and other diseases involve dis- regulation of BMP signaling, so understanding the regulation of BMP signaling is of fundamental importance. Many studies have focused on the mechanisms by which secreted BMPs and their antagonists interact to form morphogen gradients that impart position information to large fields of cells, for example in dorsoventral patterning of vertebrate and insect embryos. But other important BMP signaling processes involve short-range interactions, for example tumor-stroma interactions in cancer, or epithelia-mesenchyme interactions in tooth development. The leech (Helobdella) embryo provides a complementary model to vertebrate and Drosophila systems most commonly used for studies of BMP signaling, because it features a relatively simple cellular geometry and stereotypical cell lineages whose fates are determined in part by short-range signaling interactions. The leech is amenable to precise and reproducible experimental manipulations, based on the microinjection of markers and genetic constructs into identified, lineage-restricted stem cells (teloblasts). Thus, with Helobdella it is possible to carry out speciic manipulations of BMP signaling and assay the results precisely in the physiologically relevant context of the intact embryo. Moreover, Helobdella is evolutionarily distant from the standard models; and understanding how BMP signaling functions in homologous processes across diverse species may reveal the range of mechanisms with which BMP signaling may operate across diverse tissues and stages in individual species (e.g., human). Studies of leech development are also of importance to the field of evolutionary development (Evo-Devo) because leeches belong to a diverse, yet under-studied super-phylum of animals known as Lophotrochozoa. The specific aims for the proposed project period are to explore the spatial regulation of BMP signaling across heterologous cell types, using the Helobdella ectoderm system. The main hypothesis to be tested is that the observed differences in spatial extent of BMP5-8 signaling between two different cell types in the embryo reflects differences in the processing of BMP pro-peptides by Furin or other members of the pro- peptide convertase subtilisin/kexin (PCSK) family of proteases, which normally process BMP pro-peptides to their mature forms. To test this hypothesis, constructs expressing signaling competent, epitope-tagged variants of wild-type and cleavage site-mutant leech BMP5-8 will be microinjected to drive its expression in specific lineages of the embryo, either alone or with various leech PCSKs. Processing of the epitope-tagged BMPs will be followed by western blot analysis, and their signaling effects will be assessed using an established set of molecular and morphological criteria. In other experiments, the spatial distribution of functional BMP5-8 will be assessed directly by visualizing signaling competent, Fluorescent Protein-labeled BMP5-8 in living embryos and/or the epitope-tagged BMP5-8 variants in fixed specimens. Finally, other factors that may affect the spatial regulation of BMP5-8 signaling will be evaluated, including possible interactions with extracellular matrix via a conserved cluster of N terminal basic amino acids, internalization BMP ligand-receptor complexes, and differences between the activity and mobility of BMP heterodimers versus homodimers. The relevance of this research to public health is that it will broaden our understanding of how TGF-beta signaling processes might be regulated during human development or disease where experimentation is not possible, and in complex vertebrate model systems that are not amenable to the type of detailed analysis that is possible in the leech embryo. In addition, this research provides a forgiving and relatively inexpensive training environment in which future physicians and medical scientists can hone experimental techniques and rigorous analytical intellectual approaches for subsequent application to more clinically relevant problems.