The overall goal of our research is to understand how cell types arise during animal development, leading to a better understanding of how defects in developmental processes might influence human disease. Our approach is to generate and mine large scale gene expression datasets to identify novel developmental mechanisms, and to then use quantitative experimental approaches to test these new hypotheses. This requires the development of new experimental genomics and imaging tools and computational approaches, and most members of the Murray Lab integrate experimental and bioinformatics in their work.
Large scale mapping of the transcriptome across cells and time
Recent and ongoing technological improvements have made it possible to study gene expression at single cell resolution across all the cells of an organism. We pioneered the use of whole-embryo time-lapse microscopy of fluorescently tagged proteins to measure expression at single cell resolution and high temporal resolution. This work led to the first large-scale database of cellular resolution embryonic expression profiles in C. elegans.
We have combined this imaging-based expression profiling with single cell RNA-sequencing to measure mRNA levels genome-wide in nearly every embryonic cell. This work identified numerous candidate cell fate regulators, and general phenomena such as the presence of widespread “multilineage priming” (expression of regulators for a specific cell type in a progenitor that also produces other cell types), and convergence of gene expression patterns for cells that are unrelated in the lineage yet adopt the same fate.
We are currently adapting this approach to test regulatory networks and understand their evolution at scale by single-cell mapping of expression and chromatin in related species, and after genetic or pharmacological perturbations.
Role of cell signaling and combinatorial regulation in cell fate specification
Anterior-posterior patterning is a critical process in embryonic development. In animals from worms to humans, the Wnt signaling pathway patterns the anterior-posterior axis. This pathway shows striking regulation across the lineage in C. elegans, with the posterior daughter of nearly every cell division showing higher Wnt pathway activity than its anterior sister (as evidenced by nuclear localization of the transcriptional co-activator beta-catenin). We and others previously showed that many genes expressed in lineages derived from these posterior daughters are targets of the Wnt pathway.
However, the regulation of genes expressed in the anterior sister cells is less well understood. Such genes could either be directly or indirectly regulated by the Wnt pathway, or by independent mechanisms. We have identified a collection of anterior-specific genes and found that most of these require Wnt pathway components for their anterior specific expression. We are currently dissecting the regulatory sequences (enhancers and promoters) controlling the expression of these Wnt-repressed genes to better understand their regulation.
A striking feature of our large-scale maps of embryonic expression is the existence of “lineally repetitive” transcription factors whose expression seems to represent lineage history rather than future cell fate. These factors are expressed in partially overlapping patterns, and often have low-penetrance phenotypes as single mutants, with double or higher order mutants required to see strong phenotypes. We are testing the hypotheses that these factors represent combinatorial lineage code that specifies cell fate, and that partial redundancy in this code facilitates developmental robustness.
Control of the developmental timing, transcriptome dynamics and robustness
In addition to identifying key cis- and trans- acting regulators that control cell fates, other fundamental aspects of transcriptional regulation in development remain unknown.
Transcription itself is a complex cellular process that is modulated at multiple levels - transcription factor and RNA polymerase II binding (initiation), polymerase CTD phosphorylation (elongation) and termination to name a few. For a single gene involved in specifying a particular cell fate at a critical time during development, all of the levels of transcriptional control have to be intricately coordinated. Because of the intricacies involved, several fate specification events have backups or redundancies built into transcription to ensure developmental robustness. We are studying how rates of zygotic transcript and mRNA turnover are controlled, and how these rates influence robustness during cell fate specification.
Extensive single cell imaging and RNA sequencing in our lab has identified the transcriptome profiles of C. elegans embryonic cells. These data suggest that appropriate mRNA concentrations of fate regulators influence developmental outcomes, and that the number of mRNA molecules required varies between genes and developmental stages. We are determining how transcript synthesis rates are controlled. We do this by using genomic and single molecule techniques to measure rates of the various steps of transcription across different cell types and lineages, and how the genomic features (TF motifs, chromatin state, etc) of a gene influence transcription rates.
Similarly, our mRNA expression data indicate that mRNA degradation rates are highly regulated between genes and cells to permit cell fate specification. Analogous to how transcription factors can bind combinatorially to promoters and enhancers to regulate transcription, RNA and protein factors can bind combinatorially to mRNA transcripts to regulate mRNA stability. This could modulate mRNA decay rates throughout development in particular cell types, lineages, or developmental stages. During the maternal-to-zygotic transition in early embryogenesis, there is widespread clearance of maternal gene products as the zygotic genome is activated. The clearance of maternal gene products is important for zygotic transcription and embryonic development, supporting the idea that the timely degradation of mRNA may play important roles in cell fate decisions. However, relatively little is known about zygotic transcript turnover and its regulation, especially on a global scale across genes and cell types. We are developing approaches to measure zygotic mRNA decay rates on a transcriptome-wide scale and elucidate mechanisms of differential mRNA decay, using a combination of genomic and genetic approaches.