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Abstract ELT-2 is the major transcription factor (TF) required for Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and then persists after hatching through the larval and adult stages. Though the sites of ELT-2 binding are characterized and the transcriptional changes that result from ELT-2 depletion are known, an intestine-specific transcriptome profile spanning developmental time has been missing. We generated this dataset by performing Fluorescence Activated Cell Sorting on intestine cells at distinct developmental stages. We analyzed this dataset in conjunction with previously conducted ELT-2 studies to evaluate the role of ELT-2 in directing the intestinal gene regulatory network through development. We found that only 33% of intestine-enriched genes in the embryo were direct targets of ELT-2 but that number increased to 75% by the L3 stage. This suggests additional TFs promote intestinal transcription especially in the embryo. Furthermore, only half of ELT-2's direct target genes were dependent on ELT-2 for their proper expression levels, and an equal proportion of those responded to elt-2 depletion with over-expression as with under-expression. That is, ELT-2 can either activate or repress direct target genes. Additionally, we observed that ELT-2 repressed its own promoter, implicating new models for its autoregulation. Together, our results illustrate that ELT-2 impacts roughly 20–50% of intestine-specific genes, that ELT-2 both positively and negatively controls its direct targets, and that the current model of the intestinal regulatory network is incomplete as the factors responsible for directing the expression of many intestinal genes remain unknown. 

The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and inCulex tarsalis—an extremely efficient vector of West Nile virus (WNV)—nonexistent. We performed single-cell RNA sequencing onCx. tarsalismidguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions ofDrosophilaandAedes aegyptimidgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells (EE), suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g.,AGO2,R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish aCx. tarsalismidgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.