Molecular recording to understand the determinants of cell fate transitions in early development

Project Summary During mammalian development, a single cell gives rise to thousands of diverse and functionally distinct cell-types. Understanding how each cell-type is determined during development is one of the central questions in biology with far-reaching consequences for human health and regenerative medicine. While much of our current understanding of how cell-fate decisions are made is based on either temporally-resolved and non-destructive methods (e.g., time-lapse microscopy) or high-throughput but destructive genomic assays (e.g., single-cell RNA-seq), a new method that allows continual observation of each cell throughout the developmental process will fill the major gaps existing in our understanding of cell-fate transitions during mammalian development. Here we propose to develop molecular recording methods that enable the concurrent, non-destructive, high-throughput measurements of past cellular events and the current cell-type. Our recent methods, DNA Typewriter and ENGRAM, use precision genome editing to record cell lineage information and key transcriptional signaling events to the cell’s genome, which are recovered along with the transcriptome at the single-cell level. During the mentored K99 phase, I will further improve our methods by increasing the lineage recording efficiency (Aim 1) and testing it in the synthetic mammalian embryo systems (Aim 2). After I transition to independence in the R00 phase, I will expand the molecular recorder platform to concurrently capture diverse key cellular events (Aim 3). As our preliminary data on DNA Typewriter and ENGRAM demonstrate, we are in a strong position to carry out described molecular recording in model development systems. We anticipate that molecular recording of lineage and key signaling events in the synthetic embryo systems will deepen our model of early mammalian development. Together, our proposal will serve as a strong foundation as I transition into my independence and continue developing a general molecular recording platform.