Transcriptomic profiling of the embryonic C. elegans intestine with single-cell resolution

At a mere 20 cells, the Caenorhabditis elegans intestine regulates metabolism, energy homeostasis, host defense, yolk production, and genetic aging, all while dynamically responding to its environment. How the intestine develops to carry out these disparate functions is unknown, and how cells differ along the length of the intestine is unclear. To address these questions, we performed single-cell RNA sequencing (scRNA-seq) on FACS-enriched intestinal cells from mixed-stage C. elegans embryos. The resulting single-cell transcriptomes of 974 cells organized into 13 clusters, suggesting a diversity of cell types and states. We used two post hoc approaches to ascribe identities to each cluster. First, genes with known developmental timing in early-, mid-, and late-stages were used to place clusters in time, and smiFISH microscopy was used to fine-tune the assignments. Second, the eight late-stage clusters were assessed for their region of origin. To assign these clusters to anatomical regions, we identified marker genes for each cluster and assessed their expression along the anterior-to-posterior length of the intestine using smiFISH microscopy. Genes associated with growth and cell division were expressed in early stages, whereas genes associated with immune responses and metabolism were expressed later. Genes associated with biotic responses and RNA metabolism were the most likely to vary across the intestine's anterior-posterior axis. Finally, perturbation of anterior-localized intestinal transcripts more robustly affected intestinal function compared to central or posterior-localized genes. Overall, this research illustrates the intrinsic heterogeneity across the 20 cells of the embryonic intestine and sets the stage for future works aimed at understanding cell-specific intestinal responses to diet and the environment.