Spatially-resolved, integrated cell state and lineage tracing to define progenitor cell dynamics in early mouse development

Project Summary: Mammalian development is a probabilistic and robust process where progenitor cells must integrate genetic, epigenetic, and environmental information to generate a complex body plan. The goal of this work is to delineate how progenitor behaviors elicit specific phenotypes in health and disease. To do so, I will leverage a technology I developed in my postdoctoral work in the Weissman lab, termed PEtracer, a prime editing-based tool that enables the 1) assessment of cell state and lineage using both imaging- and sequencing- based profiling methods, including spatially-resolved techniques, 2) precise perturbation of genomes for testing hypotheses about gene function, 3) studying these processes at scale in complex tissues, in 4) a manner that minimally affects normal cell function. To showcase the PEtracer system, I used it to study a xenograft model of breast cancer where I reconstructed the 3-dimensional growth patterns of a primary tumor and then uncovered how each cell’s interactions with its neighbors and microenvironment influenced its propensity to seed metastases. These important discoveries break novel ground in cancer biology and stand to inform therapeutic development. Here, I propose to leverage this technology in engineered mouse embryonic stem cells and mouse models to create the first integrated maps of cell state and lineage during normal development, biological ciphers that could be used to understand how tissues are assembled and maintained and how these processes go awry in disease. I will use in vitro stem cell-based gastruloid models where high replication power enables a statistical view of developmental processes and allows for perturbation studies to probe the genetic determinants of cell state and lineage decisions in development. I will also use mouse models to study these questions in the complete context of a developing organism. In the K99 training phase, I will develop expertise in stem cell biology, learn how to engineer mouse models, and sharpen computational skills needed to analyze the unique datasets these projects generate. I will learn these technical skills from my brilliant co-mentors, Dr. Jonathan Weissman and Dr. Zachary Smith, and my pioneering collaborators. Further, I will gain critical experience managing a growing research program during this time. The stem cell and mouse models I engineer during my K99 phase will be foundational for my R00 phase and beyond where I will study progenitor cell dynamics during mammalian development using both cutting-edge in vitro and in vivo model systems. I aim to study how the behaviors and histories of single cells lead to complex cell states and characteristics. This work will provide a foundation for my future research program as an independent investigator at a major research institution where I will use the spatially-resolved cell state and lineage maps of progenitor cellular dynamics in normal development that I generate through this K99/R00 award to understand how these same processes go awry in developmental disorders and diseases. This work that may unlock a new era of quantitative genomics in developmental biology and guide future translational research.