The C. elegans somatic gonad was the first organ to have its cell lineage determined and the gonadal lineages of the two sexes differ greatly in their pattern of cell divisions, cell migration and cell types. Despite much study, the genetic pathways that direct early gonadal development and establish its sexual dimorphism remain largely unknown, with just a handful of regulatory genes identified from genetic screens. To help define the genetic networks that regulate gonadal development in both sexes, I employed cell-specific RNA-seq. Single sex populations of animals were generated using sex determination pathway mutants and a highly specific GFP reporter was used to isolate the somatic gonadal precursor cells, Z1/Z4 or the Z1/Z4 daughter cells, by FACS of dissociated larval cells from synchronized animals harvested just before and just after the division of Z1/Z4, when somatic gonad sexual differentiation begins. I identified transcripts present in each sex in Z1/Z4 or Z1/Z4-daughter cells and for comparison I also identified the transcripts in each sex in whole animals at both time points. Pairwise comparisons of samples identified several hundred gonad-enriched transcripts including most known Z1/Z4-enriched mRNAs, and reporter analysis has confirmed the effectiveness of this approach. Prior to the Z1/Z4 division only about 25 sex-enriched Z1/Z4 transcripts were detectable, but less than six hours later during development, I identified about 250 sex-enriched transcripts in the Z1/Z4-daughters, of which about a third also were enriched in the somatic gonad cells compared to whole animal. This blossoming of sex-specific gene expression indicates that a robust sex-specific developmental program, some of it gonad-specific, initiates in these cells around the time of the first Z1/Z4 division. Transcripts that have similar patterns in timing and expression are likely to be regulated by a similar mechanism. Sex-enriched gonadal transcripts were categorized based on similar enrichment patterns from the aforementioned pairwise comparisons. Current work is underway to identify and experimentally test shared motifs in conserved regions of promoters of transcripts that belong to the same expression category. This method of cell-specific RNA-seq should not only help define the regulatory networks controlling gonadal development but will also provide an effective general approach to identify genetic networks driving development of other cell lineages in C. elegans.
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