Transcriptional Control of Cell Specification and Differentiation
- Jeffrey A. Farrell, PhD, Stadtman Investigator, Head, Unit on Cell Specification and Differentiation
- Morgan Prochaska, PhD, Research Specialist III
- Michael Nunneley, PhD, Postdoctoral Intramural Research Training Award Fellow
- Gilseung Park, PhD, Postdoctoral Visiting Fellow
- Abhinav Sur, PhD, Postdoctoral Visiting Fellow
- Sean Lee, BS, Postbaccalaureate Intramural Research Training Award Fellow
- Avani Modak, BS, Postbaccalaureate Intramural Research Training Award Fellow
Animals consist of a collection of cells with diverse shapes, structures, and functions, a diversity that is rebuilt from scratch by every embryo. The genetic programs that direct the process are the central mystery of developmental and regenerative biology. We are interested in how decisions about the cell type to adopt are controlled, and how 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 high-temporal-resolution, single-cell RNA–sequencing approaches to identify transcriptional trajectories, i.e., the ‘highways’ or the most likely paths through gene expression that cells take during development [Farrell JA et al, Science 2018;360:eaar3131; Siebert S et al, Science 2019;365:eaav9314]. From such data, we were able to identify the sequence of genes expressed by individual cell types during early development, which 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. Work in the lab focuses on more deeply exploring such processes, using the approaches we developed. Our lab combines single-cell genomics with imaging, genetic, and classical embryological approaches to investigate the genetic control of cell specification and differentiation during vertebrate embryogenesis. We focus on zebrafish (Danio rerio) embryos as a model system in which to study these questions, because, among vertebrates, they are easy to culture, image, and manipulate, both embryologically and genetically.
Transcriptional diversity during zebrafish development
A critical step toward understanding the genetic programs that control cell specification and cell differentiation is to identify the different transcriptional cell types that are present during development, the genes expressed in each, and how their gene expression programs change over time in development. To this end, we generated a single-cell RNA-seq atlas spanning embryogenesis and early larval stages (62 timepoints from 3–120 hours post-fertilization), annotated over 300 cell types, and built a web portal (Daniocell) to enable other investigators to browse these data [Reference 3]. To better understand how cells acquire their cell type–specific features, we built a catalog of shared gene-expression programs that are re-used across many different tissues during development. We also identified transcriptional populations that are present for unusually long durations during development. We also performed focused analyses within a subset of tissues and uncovered the transcriptional profiles of several poorly characterized or unknown cell populations, including the pneumatic duct, distinct transcriptional subtypes of pericytes (a type of peri-vascular cell), best4+ cells within the intestinal epithelium, and several layers of intestinal smooth muscle. We built developmental trajectories to describe the sequence of gene expression that likely gives rise to these cell types, and we used these trajectories to determine candidate regulatory factors that may drive their specification and which can be tested in future work.
In a separate project, in collaboration with the lab of Michal Rabani, we aimed to determine whether cell type–specific gene expression patterns could emerge from regulation of mRNA destruction as well as transcription. To do so, we investigated the destruction of maternally provided mRNAs that run early development, many of which are degraded at the maternal-to-zygotic transition, when the zygotic genome activates and cells begin transcribing their own mRNA. We combined 4sU RNA labeling with single-cell RNA-seq, enabling us to distinguish between maternally provided mRNA (which is unlabeled) and zygotically produced mRNA (which incorporates the 4sU label that we provide after fertilization) [Reference 4]. By profiling development at several time points, we built kinetic models that allow comparison of rates of maternal mRNA destruction between different genes and also between different cell types. Rates of destruction of maternal mRNAs vary widely, with transcription rates often being matched to produce constant mRNA levels. Maternal mRNA stability has long been known to differ in the primordial germ cells; most early germ cell–specific mRNAs are maternally deposited and specifically stabilized in that cell type. However, we found evidence that some maternal mRNAs are also stabilized in the enveloping layer, another cell type that differentiates early in zebrafish development.
Development and function of best4+ cells
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, where we and others recently identified homologs of best4+ cells. We identified that zebrafish and human best4+ cells have similar location, regionalization, and gene expression [Reference 3]. Zebrafish’s advantages are: (1) their intestines are functionally similar to 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.
Consequences of heterogeneous developmental trajectories
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 [Reference 1]; at such boundaries, some cells switch from one specification state to another. We use the axial mesoderm as a model and seek to understand: (1) what drives cell-type switching; (2) the long-term consequences for a cell that switched; and (3) the mechanisms that assist in successful switching.
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 recently observed that cells that experience DNA damage in early zebrafish embryos initiate an unusual transcriptional response during a very limited window in development [Satija et al. Nat Biotechnol 2015 May;33:495]. Moreover, most damaged cells are not eliminated but appear to be excluded from contributing to some tissues in the animal, which suggests that responding to DNA damage may affect cells’ choices during development and which raises the question as to how that occurs. We are investigating: (1) the fate of cells in early development that experience DNA damage; (2) the role this unusual transcriptional response plays; and (3) what drives the bias in damaged cells’ future cell type.
Publications
- Farrell JA, Wang Y, Riesenfeld SJ, Shekhar K, Regev A, Schier AF. Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. Science 2018 360:eaar3131.
- Siebert S, Farrell JA, Cazet J, Abeykoon Y, Primack A, Schnitzler C, Juliano CE. Stem cell differentiation trajectories in Hydra resolved at single-cell resolution. Science 2019 365:eaav9314.
- Sur A, Wang Y, Capar P, Margolin G, Farrell JA. Single-cell analysis of shared signatures and transcriptional diversity during zebrafish development. bioRxiv 2023 10.1101/2023.03.20.533545.
- Fishman L, Nechooshtan G, Erhard F, Revev A, Farrell JA, Rabani M. Single-cell temporal dynamics reveals the relative contributions of transcription and degradation to cell-type specific gene expression in zebrafish embryos. bioRxiv 2023 10.1101/2023.04.20.537620.
- Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat Biotech 2015 33:495–502.
Collaborators
- James Gagnon, PhD, University of Utah, Salt Lake City, UT
- Celina Juliano, PhD, University of California Davis, Davis, CA
- Michal Rabani, PhD, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel
Contact
For more information, email jeffrey.farrell@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/farrell.
