Transcriptional Control 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. We published a single-cell RNA-seq (scRNA-seq) atlas of wild-type development in zebrafish, spanning the first five days of development. We created a heavily-used public resource with these data (Daniocell) that is used by investigators around the world to browse and analyze our data for their own specific research questions [Reference 2]. Our analysis of these data identified: (1) gene expression programs that are shared across cell types during development (including a novel pan-epithelial transcriptional module); (2) the molecular profiles of tissues that were previously only identified morphologically (the pneumatic duct and individual layers of intestinal smooth muscle); and (3) new transcriptional cell types, including pericyte subtypes and best4⁺ intestinal epithelial cells that resemble a population recently described in humans.

Additionally, while developmental trajectories identify the gene expression cascades that accompany the specification and differentiation of individual cell types, interpreting them biologically remains difficult. In a separate project, we helped develop MIMIR (Module Identification via Multi-source Integration for Reconstruction differentiation) to identify gene modules that correspond to biological processes from scRNA-seq trajectories by combining two info sources: similarity in expression dynamics and functional annotations [Reference 3]. Using MIMIR, we catalogued dozens of biological processes during specification and differentiation of two embryonic structures with a shared progenitor: the notochord and prechordal plate, finding known and new biological processes in these tissues and discovering new gene associations. The approach revealed the anticipatory stimulation of the unfolded protein response (UPR) prior to cargo secretion. We found that both cell types express a shared ‘core’ UPR but also ‘cargo-specific’ UPRs that differ between tissues. To investigate whether this shared expression program has shared regulatory underpinnings in both tissues, we profiled embryos with loss-of-function and gain-of-function for UPR transcription factors (TFs). We found that even though there is a shared, ‘core’ UPR, those genes are actually activated by different UPR TFs in the two tissues. Additionally, in each tissue, the same TFs that activate the ‘core’ UPR also activate the ‘cargo-specific’ UPR genes. Moreover, for xbp1 (a UPR TF) to fully activate the ‘gland-like’ cargo-specific UPR response in the hatching gland requires the developmentally programmed expression of a co-factor, bhlha15/mist1. The study provides an approach to uncover biology in scRNA-seq data from other cell types and tissues, offers a comprehensive view of cellular processes in notochord and prechordal plate development, and sheds light on cell type–specific regulation of shared processes.

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. In a collaboration with the lab of Michal Rabani, we combined 4sU RNA labeling and scRNA-seq, and we were able to distinguish between maternally loaded and zygotically transcribed mRNA in zebrafish embryos in a cell type–specific manner [Reference 4]. 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. Many cell type–specific transcripts in primordial germ cells (PGCs) are ubiquitously provided with maternal mRNAs that are stabilized in the PGCs and destroyed elsewhere. We found that differential degradation of maternal mRNAs contributes to cell type–specific gene expression programs also in enveloping layer (EVL) cells. We also identified putative sequence elements in their 3′ UTRs that may mediate their different rates of destruction in EVL cells. Future work is planned to identify the mechanisms that mediate this differential stability, and to extend labeling efforts to profile changes in stability at additional developmental stages.

best4+ cells are a recently identified (2019) epithelial population in human intestines with undefined function and developmental program. The cells are dysregulated in disease: they are depleted in inflammatory bowel disease and their characteristic genes are upregulated in colorectal cancer patients. However, it is unclear whether these changes are causes or consequences of disease. We will use zebrafish as a model, in which we and others recently identified homologs of best4⁺ cells [Reference 2]. We found that zebrafish and human best4⁺ cells have similar location, regionalization, and gene expression. Zebrafish’s advantages are that: (1) their intestines are functionally similar to those of mammals but optically clear, facilitating experimental observation; (2) best4⁺ cells are missing in popular rodent intestinal models; (3) experiments can be performed in vivo, in regionalized intestines with surrounding mesenchyme and smooth muscle, which produce many of the key signals that regulate the intestinal epithelium. We aim to: (1) determine the effects on the intestine of best4⁺ cell loss by genetically ablating best4⁺ cells; (2) determine the effects of inflammation on best4⁺ cells; (3) build a gene-regulatory network (GRN) of intestinal epithelial specification; and (4) determine the developmental program that specifies best4⁺ cells. Our results will help identify the function and role in disease of best4⁺ cells and suggest how to modulate best4⁺ cell function or cell number to potentially treat disease.

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 (Hedgehog) signaling and inhibitory BMP (bone morphogenetic protein) signaling drives iSMC layer specification sequentially, and cells are then oriented by mechanical forces, i.e., strain from the gut orients cells in the inner layer, and then inner layer contractions orient outer layer cells perpendicularly. We recently identified six zebrafish iMese/iSMC transcriptional populations [Reference 2], but it is unclear how those transcriptional differences interact with biomechanical inputs, i.e., whether one drives the other or whether 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.

Distinct cell types can arise through many developmental trajectories or developmental histories. We and others have observed refinement at the boundaries between groups of cells specified to become different tissues, where some cells initially exhibit a hybrid state characterized by gene expression consistent with multiple cell types [Reference 1]. We use the axial mesoderm as a model and seek to understand: (1) how is this hybrid expression generated; (2) the states and fates cells adopt downstream of a hybrid identity; (3) the long-term consequences for a cell that experienced a hybrid identity; and (4) the mechanisms that assist in successful resolution of hybrid gene expression states.

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 recently observed that cells that experience DNA damage in early zebrafish embryos initiate an unusual transcriptional response during a very limited window in development [Reference 5]. Moreover, most damaged cells are not eliminated but appear to be excluded from contributing to some tissues in the animal, suggesting 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; and (3) the mechanism that drives the observed bias in cell fate among damaged cells.