Project summary Bone Morphogenetic Proteins (BMPs) function as morphogens during development to specify spatial organization of embryonic axes and organ structures. Numerous extracellular antagonists and co-factors work in concert to dynamically control BMP signaling distributions along the dorsal-ventral (DV) embryonic axis in vertebrates to induce space and time-dependent patterns of gene expression. As the embryo progresses from the blastula stage into the gastrula stage, zygotic feedback in response to BMP signaling begins and cells start to play an active role by simultaneously responding to BMPs and regulating the secretion of antagonists and other factors that shape the BMP gradient. However it is not well understood how the system dynamically regulates pattern formation. Studies of gastrula stage BMP pattern formation provide an ideal context to unravel how feedback regulation shapes the gradients of BMP signaling in a vertebrate system. We hypothesize that BMP-mediated feedback by admp, sizzled, tld, and bambi dynamically shape the gradient in gastrula embryos. Our objective is to delineate how coupled patterning and feedback shape gradients and discover mechanisms of BMP regulation in zebrafish gastrula. In Aim 1 we will quantify spatiotemporal BMP signaling and feedback target gene expression in wild-type and mutant zebrafish gastrula embryos. Furthermore, we will measure phospho-Smad 1/5 (PSmad) levels in toto in wild-type (wt) gastrula embryos, and develop our new wavelet-based segmentation and image analysis software WaveSeg (https://waveletseg.weebly.com) to quantify, segment, and register the data. This work will provide the first- ever quantitative data needed to identify feedback relationships that shape the PSmad gradient. Quantification of BMP patterning in gastrula stage embryos will provide insight into the role of feedback in gradient refinement in a rapidly changing spatial domain. In Aim 2 we will develop a 3-dimensional growing mesh finite element model of BMP pattern formation in gastrula stage embryos. Evaluation of the models against quantitative imaging data provides a rigorous basis to discern the mechanisms of gastrula embryo pattern formation and generate new tests to validate or refute the consistent mechanisms. The seamless integration and back-and- forth between imaging, genetics, and modeling will elucidate how the extracellular BMP pathway factors modulate the BMP signaling gradient. Lastly, in Aim 3 we will investigate the feedback loops in BMP signaling that have been proposed to provide embryonic scale invariance, robustness with respect to partial loss of network components, and increase the dynamic range of signal interpretation. Understanding these networks of BMP regulation will provide insight into BMP function in other contexts including organogenesis, cancer metastasis, angiogenesis and development.

Public Health Relevance The proposed work will determine how Bone Morphogenetic Proteins (BMPs) are dynamically controlled during development. Elucidation of BMP function is of paramount importance due to the genes' roles in organogenesis, cancer metastasis, angiogenesis and development. Quantitative imaging of gene expression and PSmad signaling and finite element modeling will elucidate the mechanisms of BMP signal regulation and pattern control.