Research Areas
Focussing on the primary step in gene expression - transcription, and investigating mechanisms of transcriptional control that drive cell fate specification.
Overview
All cells have to integrate signals from the environment and make decisions on dealing with these external cues. The developing embryo allows us to explore how decision making happens across multiple different cells all at once.
As a single-celled zygote divides and differentiates, what factors are necessary to ‘tell’ each cell whether they are destined to become muscle, skin or the myriad of other cell types in the body? Gene expression programs are fundamental to these cell fate decisions. Our lab focuses on the primary step in gene expression, transcription and investigates mechanisms of transcriptional control that drive cell fate specification.
We use the microscopic worm, Caenorhabditis elegans to address these questions. The C. elegans embryo develops through an invariant lineage—wild-type embryos follow a stereotyped pattern of cell divisions giving rise to the exact set of 558 cells at the end of embryogenesis. The identities and lineage histories of each of these cells are very well known, providing a beautiful system to study what happens to individual cell fates when transcriptional programs are perturbed.
Confocal image of a C. elegans embryo. Nuclei are marked with histone::mCherry and GFP from an RNA polymerase II reporter
Focus 1
Identifying the determinants that regulate transcription rate and mRNA output
Transcription occurs through multiple intricately regulated steps. Yet, it is still unknown which steps and to what extent each impacts transcription rate.
Based on preliminary data, three aspects are likely important to achieve the appropriate rate and mRNA dosage.
This lab will work toward determining the contributions of:
transcription factories
transcription initiation
elongation, in regulating transcription rate and dosage
Shown here are two embryonic precursor cells destined to form muscle (left) and intestinal (right) tissue. Transcription by RNA polymerase II (crab claw structure) is essential for cell fate decisions and we aim to understand all of the steps that are responsible for this.
Focus 2
Establishing the connection between transcription kinetics and mRNA dosage precision
RNA levels are affected by transcription noise.
The relationship between the transcription kinetics and transcriptional precision is not well understood.
Further, mechanisms that reduce transcription noise and support the highly invariant development of the C. elegans embryo are unknown.
We are combining live RNA imaging with automated lineage analysis to directly link transcription dynamics with single cell decisions.
Live transcripts will be visualized at single loci using the MS2-MCP system. When these dynamics are perturbed in mutants compared to wild-type (WT), cell fates can be disrupted (Xs on the lineage tree)
Focus 3
Connecting transcriptional dsyregulation and variable expressivity in developmental disorders
Our estimates of genome-wide transcript accumulation across stages in the early C. elegans embryo determined that rates were highest at the 8-cell stage, soon after the onset of zygotic transcription and when cell sizes are relatively larger. Cell size-dependent scaling of mRNA quantities has been reported in multiple organisms, although the factors involved in scaling are not completely known.
Using RNAi and mutants that increase or decrease cell size, we will investigate the dependence of mRNA quantities on the size of cells undergoing cell fate decisions.
How does the cell ‘know’ its relative size, and scale metabolic processess accordingly?
Focus 4
C. elegans as a model to study transcriptional dysregulation and variable expressivity in developmental disorders
The ability to correlate molecular changes in individual cells with whole embryo defects provides an exciting opportunity to model human developmental disorders of transcription in the worm embryo.
Disorders caused by mutations in genes encoding the basal transcription machinery have common phenotypes, but molecular basis for transcription-associated developmental disorders are unclear.
In collaboration with the OMICS initiative at the Children’s Hospital of Philadelphia, our lab will create worm models of pathogenic variants or variants of uncertain significance in transcriptional component genes.
We will use CRISPR editing to introduce relevant mutations into the worm ortholog of a human disease gene (or create a humanized worm by replacing the C. elegans ortholog with a human gene). These disease model worms will be used to characterize transcriptome changes (single locus imaging and global RNA sequencing) and cell fate defects using automated lineage analysis techniques.