Supplementary Figure 1: Additional examples of tissue-specific CRISPR mutagenesis in wing disc and abdomen. (A) CRISPR mutagenesis of smo in the posterior compartment of the wing imaginal disc. Smo protein was detected by immunohistochemistry. Smo is normally expressed in all wing disc cells, but protein levels are higher in the posterior compartment (see Control (hh-Gal4 UAS-cas9.P2)). In hh-Gal4 UAS-cas9.P2 pCFD6-smo 2x wing discs cells in the posterior compartment express no or reduced levels of Smo, presumably reflecting cells containing only one or no functional smo alleles. (B) CRISPR mutagenesis of sens in the dorsal compartment of wing imaginal discs with ap-Gal4 leads to a loss of Sens expression in most, but not all cells. (C) Mutagenesis of y in the dorsal abdomen.In pnr-Gal4 UAS-cas9.P2 pCFD6-y 2x animals cuticle coloration is uniformly changed in a broad stripe centred around the dorsal midline, compared to control animals ). Note that the strong phenotype mediated by pCFD6-y 2x is in line with the high levels of mutagenesis with this construct reported in Figure 4a. Control hh-Gal4 UAS-cas9.P2 pCFD6-smo 2x

Epithelia are exposed to diverse types of stress and damage from pathogens and the environment, and respond by regenerating. Yet, the proximal mechanisms that sense epithelial damage remain poorly understood. Here we report that p38 signaling is activated in adult Drosophila midgut enterocytes in response to diverse stresses including pathogenic bacterial infection and chemical and mechanical insult. Two upstream kinases, Ask1 and Licorne (MKK3), are required for p38 activation following infection, oxidative stress, detergent exposure and wounding. Ask1-p38 signaling in enterocytes is required upon infection to promote full intestinal stem cell (ISC) activation and regeneration, partly through Upd3/Jak-Stat signaling. Furthermore, reactive oxygen species (ROS) produced by the NADPH oxidase Nox in enterocytes, are required for p38 activation in enterocytes following infection or wounding, and for ISC activation upon infection or detergent exposure. We propose that Nox-ROS-Ask1-MKK3-p38 signaling in enterocytes integrates multiple different stresses to induce regeneration.

SummaryIntestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.