PROJECT ABSTRACT The long-term goal of this project is to understand how embryos accurately transmit instructions to their constituent cells. This proposal focuses on signaling by Nodal, a model morphogen that induces endoderm and mesoderm formation in early vertebrate embryos. Genetic and biochemical studies demonstrate that target cells select fates based on their position a gradient of Nodal signaling activity. However, the mechanisms of gradient formation and signal interpretation remain unclear. The proposed experiments will test competing mechanisms for the spatial spread of signaling from an endogenous Nodal source (Aim 1), and explain how similar signaling levels can specify more than one cell fate (Aim 2). These studies will be carried out in zebrafish to take advantage of its unique genetic and optical tractability. This work will resolve controversies concerning the role of diffusion in transporting endogenous Nodal ligands (K99), explore whether stochastic noise in Nodal signaling influences germ layer specification in a vertebrate embryo (K99, R00) and identify new Nodal-dependent gene expression programs (R00). Clarifying how Nodal signaling functions in vivo may also have implications for medicine, as misregulation of Nodal signaling can drive birth defects and cancer in humans. Finally, understanding the mechanisms that allow Nodal signals to transmit precise instructions will inform tissue engineering and regenerative biology, as Nodal is used to control cell differentiation in vitro. My career goal is to run an academic lab that studies how developing embryos achieve precise patterning in the face of unexpected perturbations. The proposed experiments will give me the experience in embryology, zebrafish genetics and optogenetics required for this goal. I have developed a detailed training plan with my mentor, Dr. Alexander Schier, to help me transition to independence. I will meet regularly with Dr. Schier to discuss research progress, strategies for grant writing, student mentorship and lab management. To practice my mentorship skills, I will oversee the work of a graduate student. To broaden my scientific network, I will present my work at two international meetings per year and participate in joint lab meetings with the Megason, Zon and Ramanathan groups at Harvard. To seek out additional mentorship, I assembled a K99 advisory committee consisting of Dr. Adam Cohen, Dr. Allon Klein, and Dr. Sean Megason. Their expertise in optogenetics, single-cell transcriptomics and quantitative imaging will help me to execute my research plan on the proposed schedule. As a member of the Harvard Department of Molecular and Cellular Biology, I will have access to leaders in developmental biology, biophysics and genetics, as well as cutting-edge core facilities. The Pathway to Independence Award will provide the time and freedom required to initiate an ambitious research program on the mechanisms of robust patterning in developing embryos.