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? How do genetic programs minimize errors to ensure robust developmental outcomes? 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, image, and manipulate both embryologically and genetically. Using zebrafish enables us to study developmental biology in vivo, where cells develop in their native context, interacting with many other cell types and tissues through signaling and mechanical force. Specific areas of investigation include: 1. Genetic Underpinnings of Cell Specification and 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. Recently, we created a heavily-used public resource (Daniocell) that profiled gene expression at the single-cell level across the first five days of zebrafish development and is used by investigators around the world to browse and query to rapidly answer questions about when and where genes are expressed. During this project period, we expanded that resource to include an interactive desktop version, DaniocellDesktop, that enables users to perform their own simple bioinformatic analyses, including redefining cell populations, performing differential expression testing, and generating publication-ready customized plots. Through collaborations, we also contributed to several projects this project period, including: (1) helping to identify the changes that occur in spermatogenesis during aging that may contribute to declining fertility with age (Sposato et al.), (2) providing unpublished data that helped determine that hair cells (the mechanosensory cells in the ear that detect sound) become transcriptionally similar to a related mechanosensory cell type (in the lateral line) when they lack the transcription factor prdm1a (Sandler et al.), and (3) helped identify the transcriptional cascade as putative precursor cells are specified and differentiate into leptomeningeal barrier cells and meningeal fibroblasts (Venero Galanternik et al.). Finally, developmental trajectories identify the gene expression cascades that accompany the specification and differentiation of individual cell types, but interpreting them biologically remains difficult. During this project period, we developed an approach (MIMIR) to organize the genes identified within a developmental trajectory into functional gene modules that help explain the biological processes that accompany the differentiation of individual cell types (Wang et al.). MIMIR integrates similarity in expression dynamics and functional annotations. We used MIMIR to catalog dozens of new and previously known differentiation processes and their dynamics during zebrafish axial mesoderm development, which included anticipatory stimulation of the unfolded protein response (UPR) prior to cargo secretion. We then profiled loss-of-function and gain-of-function embryos to determine the regulatory logic underlying the UPR activation and identified that a shared, core program is activated by different TFs in different cell types, that single TFs activate both the core UPR and cargo-specific UPR responses in each cell type, and that the hatching gland UPR requires the cell-type-specific co-factor, mist1. 2. Differences in RNA stability during development Cell specification and differentiation during development is ultimately regulated by cell-type-specific transcriptional states that drive their different behavior. Many efforts to understand the gene regulatory network that generates cell-type-specific transcriptional states focuses on differences in RNA production, but equally important in determining cellular transcriptional states are rates of RNA destruction. Previously, in a collaboration with the lab of Michal Rabani, we combined 4sU RNA labeling and single-cell RNAseq and were able to distinguish between maternally loaded and zygotically transcribed mRNA in zebrafish embryos in a cell-type-specific manner. This allowed us to quantify the rates of destruction of maternally loaded mRNAs during the maternal-to-zygotic transition using new kinetic models developed by the Rabani lab. Ongoing work is underway to identify the mechanisms that mediate differential stability of maternal mRNAs in enveloping layer cells, to extend labeling efforts to profile changes in stability at additional developmental stages, and to develop more sophisticated and low-cost methods for combining 4sU RNA labeling with scRNAseq. 3. Development and function of best4+ intestinal cells best4+ intestinal epithelial cells were first molecularly characterized in 2019 in humans and named for one of their many cell-type-specific genes. best4+ cells are pH-responsive and uniquely express several intestinal hormones and receptors, carbonic anhydrases, and ion transporters, suggesting they may monitor and coordinate gut behavior, respond to luminal pH, and have key roles in fluid homeostasis, diarrheal flushing of pathogens, and mucous layer integrity. best4+ cell dysregulation may contribute to disease, since best4+ cells are depleted in inflammatory bowel disease and potentially increased in colorectal cancer. However, it is unclear whether these changes contribute to or result from disease. Moreover, the developmental program of best4+ cells remain unknown in any animal. We and others recently discovered an analogous cell population in the zebrafish gut with similar cell-type-specific gene expression. We have initiated a project to use zebrafish to (1) determine the developmental program that specifies best4+ cells, (2) identify functional roles for best4+ cells, and (3) determine whether best4+ cell loss contributes to or results from intestinal disease. 4. Development and function of intestinal smooth muscle and mesenchyme The gut depends on support from surrounding intestinal smooth muscle cells (iSMCs) and intestinal mesenchyme (iMese) to drive peristalsis and provide secreted signals that control intestinal proliferation and differentiation. However, many aspects of the development of this crucial tissue are not yet understood in any animal. In chick, interplay between stimulatory Hh signaling and inhibitory BMP signaling drives iSMC layer specification sequentially, and cells are then oriented by mechanical forces—strain from the gut orients cells in the inner layer, and then inner layer contractions orient outer layer cells perpendicularly. We recently identified 6 zebrafish iMese/iSMC transcriptional populations, but it is unclear how those transcriptional differences interact with biomechanical inputs — does one drive the other, or is there an interplay between both processes? This project aims to (1) characterize each iMese/iSMC cell population and (2) identify how signaling, mechanical inputs, contractile force, and transcription factors cooperatively build iSMC/iMese gene expression programs. 5. 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 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? 6. 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 and potentially other stresses during early blastula stages initiate a tp53-dependent transcriptional response that includes developmental regulators as well as canonical stress response genes. We have also observed that this response is not evenly distributed across the blastula, but is significantly enriched on the ventral side. The goal of this project is to understand: (1) what stressors occur naturally in blastula-stage embryos that trigger this response, (2) whether the induced developmental regulators have a role in the DNA damage response, (3) why the response is distributed unevenly across the embryo, (4) whether there is an interaction between developmental pathways and stress response pathways in the early embryo, and (5) whether that interaction is important for proper development.