PROJECT SUMMARY Disruption of developmental processes, often due to genetic mutation, can lead to structural birth defects that have life-long consequences for affected individuals and their families. Identification of patient- specific genetic changes, followed by mechanistic studies, can increase our understanding of how developmental perturbations lead to defects and offer insight into potential future clinical interventions. Our long-term goal is to identify molecular and cellular functions of patient-identified, disorder-associated genes with unknown roles in embryonic development. Here, we focus on one such gene, Mannose- Binding Lectin-Associated Serine Protease 1 (Masp1), mutations of which are associated with the developmental disorder 3MC Syndrome. Our objective for this proposal is to identify the underlying molecular functions of Masp1 in development. We have found that genetic perturbations of Masp1 expression (both overexpression and knockdown) in African Clawed Frog (Xenopus laevis) embryos leads to complementary changes in dorsal anterior structures such as altered head size and cement gland size and shape. Additionally, we found that Masp1 manipulation leads to altered gene expression patterns suggestive of embryonic patterning defects that could disrupt ectodermal-derived tissues, possibly through Masp1 intersection with the BMP signaling pathway. In this project, we will continue to use Xenopus laevis as a model system to address our central hypothesis that Masp1 is involved in ectoderm patterning through positive regulation of the BMP signaling gradient. First, we will perform targeted genetic perturbations of Masp1 followed by phenotypic and gene expression analysis to identify a role of Masp1 proteolytic cleavage in tissue patterning during gastrulation (Aim 1). Next, we will use both targeted (in vivo luciferase reporter assay) and unbiased (co-immunoprecipitation followed by LC/MS) approaches to identify Masp1 targets and determine how Masp1 regulates BMP signaling (Aim 2). Through this work, we will provide undergraduate and graduate students with meaningful research experiences in the field of developmental biology. Upon completion of this study, we expect to determine the mechanistic basis for Masp1 involvement in early development and identify a new player in regulation of BMP morphogenic signaling with future implications for interventions of Masp1- and BMP-associated developmental disorders.