General approach for in vivo recovery of cell type-specific effector gene sets

Abstract

Differentially expressed, cell type-specific effector gene sets hold the key to multiple important problems in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various practical applications. Although individual cell types of interest have been recovered by various methods and analyzed, systematic recovery of multiple cell type-specific gene sets from whole developing organisms has remained problematic. Here we describe a general methodology using the sea urchin embryo, a material of choice because of the large-scale GRNs already solved for this model system. This method utilizes the regulatory states expressed by given cells of the embryo to define cell type and includes a fluorescence activated cell sorting (FACS) procedure that results in no perturbation of transcript representation. We have extensively validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated embryonic skeletogenic cells and as a consequence, generated a prototypical cell type-specific transcriptome database.

Genome-wide assessment of differential effector gene use in embryogenesis

“…S3 (see legend for identities of these transcripts). The result is clear; exactly as in the preceding study on transcripts enriched in skeletogenic cells (Barsi et al, 2014), transcript sets isolated by this method are, by direct observation, specifically expressed in the expected spatial domains. FACS isolation of the cells expressing the respective regulatory genes thus effectively worked to separate out these embryonic cell populations, irrespective of the very distinct cell types and embryonic locations sampled.…”

“…This was done in extenso in the preceding study on skeletogenic cells (Barsi et al, 2014). In that study, virtually every one of a large number of genes indicated by transcriptome analysis of the presumed skeletogenic population to be specifically expressed was indeed shown by in situ hybridization to be transcribed in skeletogenic cells.…”

“…They were identified by sorting on the GFP generated by a recombineered lhx2/ 9-GFP bacterial artificial chromosome (BAC). (3) Presumptive skeletogenic cells were identified as cells expressing tbrain or alx1 recombinant BACs at 24 hpf, as described at length earlier (Barsi et al, 2014). (4) Cells of the stomodeal region of the oral ectoderm were identified by expression of gsc at 35 hpf (Li et al, 2013).…”

“…Using a method pioneered in our laboratory, we exploit known regulatory states to mark cell types of interest fluorescently and separate them out by way of flow cytometry (akin to Defaye and Perrin, 2014;Barsi et al, 2014). Specifically, we use fluorescenceactivated cell sorting (FACS) to separate out six different populations of cells from the pregastrular and early gastrula sea urchin embryo.…”

Genome-wide identification of enhancer elements

“…New quantitative transcriptome methods have enabled us to define the set of all factors relevant to a given network (Barsi et al, 2014;Barsi, Tu, et al, 2015;Fischer et al, 2014;Tulin et al, 2013). However, once the players are known, researchers must navigate a low-throughput bottleneck when searching for active modules regulating network genes.…”