Barsi Lab

Identify genes that collectively endow each cell type with a unique set of properties

ŸBarsi et al. 2015 Development. 142(22):3892-901

ŸBarsi et al. 2014 Genome Research. 24(5):860-8

Transcriptome profiling provides the parts list: a catalog of all genes expressed during a particular developmental stage of the embryo. Integrating across time, we obtain the transcriptional dynamics for each gene. However, quantifying transcripts in this manner has its fallacy because it reflects an average across all cell types. That is to say, not all genes are expressed in all cells. An album of gene expression patterns obtained through RNA in situ hybridization could in theory, resolve the problem. Nevertheless, this is impractical when dealing with an entire transcriptome. Alternatively, it is possible to select transcription factors that serve as cell specific markers. Genetically engineering an artificial chromosome to usurp the cis-regulatory apparatus of the marker gene, allows us to generate transgenic embryos for the purpose of fluorescently labeling cell types of interest. Embryonic disaggregation followed by FACS can ensure the recovery of ample material for downstream applications, comparable to quantities typically obtained from tissue culture. Exactly which cell types may be recovered in this manner, largely depends on the degree to which their regulatory state (combination of transcription factors within the nucleus) is known. For those cells isolated in this manner, deep transcriptome sequencing would provide, for each time point assessed, a comprehensive catalog of gene expression unique to that cell type. If then, we were to compare the transcriptome from an individual cell type to one derived from the assorted cells that constitute the non-fluorescent cell population, it is possible to distinguish all genes differentially expressed in the cell type of interest. This is referred to as the Effector Gene Cohort. Thus, we are able to itemize the components that collectively endow a cell of its unique properties.

Explain the mechanism by which cells acquire their fate and organize in space and time

ŸBarsi et al. 2016 Developmental Biology. 409(1):310-8

ŸBarsi et al. 2015 Development. 142(5):953-61

Gene Regulatory Networks (GRNs) are graphical models that depict the cascade of interactions between transcription factors and the genes they regulate. In the context of developmental biology, a GRN reveals the steps necessary for a particular developmental process to occur. The first step is to identify all of the transcription factors involved, then perturb each one in turn and quantify what effect, if any, this has on the transcription of all others. From such experiments, we can infer the relationship between these genes. Ultimately, each transcription factor-DNA interaction is authenticated by cis-regulatory analysis in order to corroborate or amend network topology. Taken together, an authenticated GRN provides a step-by-step explanation of the regulatory events driving pattern formation within an embryo.

Develop novel technology to visualize gene expression with unprecedented precision

ŸChoi….Barsi….Pierce. 2016 Development. 143(19):3632-3637

The programmable chemistry of nucleic acid base-pairing can be harnessed to perform a variety of un-natural tasks. Given the advanced state of current confocal microscopy, we are able visualize multiple color channels simultaneously. However, traditional RNA in situ hybridization techniques expose biological specimens to enzymatic reagents repetitively in order to visualize multiple targets. This approach degrades specimen integrity and adversely affects spatial resolution. We demonstrate an orthogonal stratagem to accomplish the same end that circumvents the use of enzymatic reagents entirely. The result is the ability to monitor transcription across multiple genes simultaneously, with unprecedented precision. Watch video

Develop novel methodology to identify cis-regulatory elements on a genome-wide scale 

ŸTulin, Barsi, Bocconcelli and Smith. 2016 Int J Dev Biol. 60(4-6):141-50

All cells from an individual animal contain the same DNA and the simplest of animals consist of many different cells. Hence, one must first ask: what mechanism enables the zygote to generate a plethora of cell-types? The answer, in general terms, is differential gene expression. The control apparatus that governs the expression of a gene is itself a sequence of DNA, though not necessarily located immediately upstream of the transcription start site, as often assumed. In fact, a survey across model organisms reveals that it is frequently fragmented into disparate control modules whose individual loci can map great distances from the gene they influence. To understand differential gene expression we must directly address this apparatus, but in order to do so we must (a) be able to identify cis-regulatory elements from the bulk of non-coding genomic sequence, and (b) associate each cis-regulatory element identified to the gene it regulates. By combining the concept of chromosome conformation capture with that of chromatin immunoprecipitation sequencing, we have come up with a way to identify active cis-regulatory elements and simultaneously distinguish the gene that each element regulates.

Explain the mechanism of intercellular Notch signal transduction within mammals

ŸWu….Barsi….Artzt. 2007 Genesis. 45(11):722-7

ŸBarsi et al. 2005 Mech Dev. 122(10):1106-17

The Notch signaling pathway is one of the most commonly activated signaling pathways in cancer. It had been extensively studied when I entered graduate school. At the time, mutagenesis screens across multiple model organisms had just about reached saturation. It is therefore surprising that we discovered a novel gene (Mindbomb) essential to this pathway while working on mouse development. We genetically engineered the first transgenic mice carrying mutations in either of the two homologs and determined that Mindbomb1’s function is required to expose the S3 cleavage site on the Notch receptor: a mechanical explanation accepted to this day of what occurs at the molecular level.