Transcriptional Control of Cell Specification and Differentiation During Zebrafish Development

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Animals consist of a collection of cells with beautifully diverse shapes, structures, and functions, and this diversity is rebuilt from scratch by every embryo. Understanding the genetic programs that direct this process is the central mystery of developmental and regenerative biology. How are decisions about what cell type to adopt controlled? What genetic programs direct the morphological and functional specialization of different cells? The single-cell revolution in developmental biology has given us new access and new tools to address these questions. I previously developed approaches to identify transcriptional trajectories from high temporal resolution single-cell RNA sequencing. These are the highways or most likely paths through gene expression that cells take during development. From these data, we were able to identify the sequence of genes expressed by individual cell types during early development. This provides insight into the genetic programs that regulate cells choice of cell type and then their downstream functional transformations at a wider breadth than was previously achievable. We combine single-cell genomics, imaging, genetic, and classical embryological approaches to investigate the genetic control of cell specification and differentiation during vertebrate embryogenesis. We focus on zebrafish embryos as a model system to study these questions, because among vertebrates, they are easy to culture, imagine, and manipulate both embryologically and genetically. Our specific areas of investigation are: 1. Genetic Underpinnings of Cell Differentiation Specified cells must acquire their particular morphology and functionality during differentiation, which is driven by cell-type-specific gene expression and often results in dramatic changes in basic cell biological processes. We aim to identify those genes that drive differentiation and understand their regulation. To do this, we are (a) generating a single-cell RNAseq atlas that spans 14120 hours post-fertilization during zebrafish embryonic development, (b) identifying and annotating the cell types therein based on known markers, (c) finding the cascade of genes expressed during differentiation in every cell type, (d) determining their membership in functional gene modules (groups of genes that work together), (e) associating them with that cell types cell biological transformations during differentiation, and (f) identifying which gene expression programs are re-used in multiple cell types. These studies will enable comparisons of differentiation gene deployment across cell types, potentially suggest roles for understudied genes based on their association with known processes, and help identify how often shared cellular traits are driven by shared gene expression programs and shared regulatory wiring during development. 2. Consequences of Heterogeneous Developmental Trajectories Distinct cell types can arise through multiple developmental trajectories or developmental histories. We and others have observed refinement at the boundaries between group of cells specified to become different tissues, where some cells will switch from one specification state to another. We use the axial mesoderm as a model and seek to understand: (1) what drives this switching, (2) what are the long-term consequences for a cell that switched, and (3) what mechanisms assist in successful switching? 3. Effect of Environmental Insults on Developmental Choices During early embryogenesis, a field of equipotent cells are instructed to initiate different gene expression programs by external developmental signals and cell intrinsic cues. We have recently observed that cells that experience DNA damage in early zebrafish embryos initiate an unusual transcriptional response during a very limited window in development. Moreover, most damaged cells are not eliminated but seem to be excluded from contributing to some tissues in the animal. This suggests that responding to DNA damage may affect cell fate decisions during early development. We are investigating: (1) the fate of cells in early development that experience DNA damage, (2) the role of DNA-damage responsive gene expression that is specific to early embryogenesis, (3) the mechanism that drives the observed bias in cell fate among damaged cells.