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. The genetic programs that direct this process are a 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 cell fate decisions 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 A central quest in developmental biology is to understand the genetic programs that confer specific identities, morphologies, and behaviors to the many different cell types in a functioning animal. Our goal is to identify the cascades of gene expression within distinct cell types that drive specification and differentiation and to understand their regulation. To do this, we have (a) generated a single-cell RNAseq atlas that spans 14-120 hours post-fertilization during zebrafish embryonic development, (b) integrated this with our previous data from 3-12 hours post-fertilization, and (c) identified and annotated the cell types therein based on known markers. Additionally, we (d) determined the co-expressed gene modules (groups of genes that work together) that are re-used in multiple cell types during development, and (e) characterized several tissues in greater depth, including reconstruction of their transcriptional developmental trajectories. This has led to the identification of previously undiscovered cell types (surfactant producing cells), revealed that some poorly characterized cell types are actually comprised of several distinct subtypes (pericytes and smooth muscle), and identified new zebrafish cell types that are homologous to understudied cell types involved in human disease (specialized intestinal enterocytes). Finally, we have (f) generated a public resource (Daniocell) that will enable investigators around the world to browse and analyze our data for their own specific research questions. 2. Resolution and Consequences of Hybrid Cell States 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 initially exhibit a hybrid state characterized by gene expression consistent with multiple cell types. We use the axial mesoderm as a model and seek to understand: (1) how is this hybrid expression generated, (2) what states and fates do cells adopt downstream of a hybrid identity, (3) what are the long-term consequences for a cell that experienced a hybrid identity, and (4) what mechanisms assist in successful resolution of hybrid gene expression states? 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.