Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, we generated RNA-sequencing transcriptomes of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, we identify transcriptional diversities among cell types and document regional differences within each cell type that define further specialization. We validate cell-specific and regional Gal4 drivers; demonstrate roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation, and study the transcriptional response of midgut cells upon infection. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions.

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-β/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.

The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. We find that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples ISC proliferation from expression of niche-derived signals but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing.

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Highlights d Oral infection is more dramatic in larvae than adults and induces developmental delay d Larval gut repair is limited and achieved by differentiation of adult gut precursors d Developmental delay allows for reconstitution of the pool of adult midgut precursors d The JAK-STAT pathway is induced by infection and controls precursor differentiation