A key goal in developmental biology is to understand how the brain is specified and organized regionally, cellularly and molecularly. Central to this vision is determining the origins and fates of cells during development, and thus map the progressive steps of cell specification and lineage divergences. Focused efforts have provided insight into specific cell types and lineages, however global views of these processes have been lacking. Recent technological breakthroughs in single-cell transcriptomics and lineage tracing using CRISPR-Cas9 tools are now enabling the realization of this vision. The long-term goal of this project is to obtain global views of cellular relationships and molecular changes during neural development and cell type diversification in the vertebrate brain. These include generating large-scale, single-cell resolution cell specification trajectories that describe molecular cascades underlying cell fate specification (Aim 1), and lineage trees that describe the history of cell divisions (Aim 2). These trees represent many key aspects of developmental decisions and can be used to determine gene expression cascades during cell specification and regulatory factors involved in progenitor priming and neuron identity (Aim 1). Furthermore, they can address how often transcriptional and lineage identities are related (Aim 2). These studies will generate resources for genome-wide and single-cell analysis of brain development and reveal cellular and molecular mechanisms for generating neuronal cell diversity. My career goal is to run an academic lab aimed at investigating cellular and molecular features underlying brain development, neurogenesis and neural stem cell activity using global and focused approaches. The proposed research draws on my previous experience with characterizing gene regulatory networks in neurogenesis and extends it to a new model organism, zebrafish, while exposing me to new experimental and analytical methods. I have developed a detailed training plan with my co-mentors, Drs. Len Zon, Alex Schier, and Josh Sanes, who have combined expertise in development, behavior, neurobiology, single-cell analysis and disease modeling. To help me transition to independence, we will meet regularly to discuss research progress, brainstorm ideas, and obtain guidance on grant writing, mentorship and lab management. My K99 advisory committee consists of Drs. Allon Klein, Sean Megason and Gord Fishell, whose collective expertise in single-cell genomics, zebrafish development, and neurobiology will provide me with technical and conceptual feedback in executing my research plan. I will continue to mentor a research technician/graduate student, will present my research at two meetings per year, take courses on bioinformatic analysis, mathematical modeling and grant writing, and attend seminars to broaden my scientific training. As a member of the Harvard Department of Molecular and Cellular Biology, I will have access to leaders in developmental biology, neurobiology and genetics, as well as cutting-edge core facilities. The Pathway to Independence Award will provide me with resources to initiate an ambitious research program and obtain additional training to maximize my chances of a successful transition to independence.
The vast diversity of cell types observed in the vertebrate brain arises from a small pool of embryonic progenitors, but the cellular and molecular details of how progenitors are progressively specified to generate this diversity at a global scale are still lacking. This project will apply single-cell RNA-sequencing and CRISPR-Cas9 lineage tracing methods to generate cell specification trajectories and lineage trees during zebrafish brain development. These datasets will reveal gene expression landscapes, progenitor dynamics and cell lineage relationships that underlie brain development, and may inform strategies for translation into disease modeling and regeneration.
