br Results and Discussion In order to

Results and Discussion In order to identify regulators of ISC behavior, we carried out a screen using esgGal4 in the posterior midgut. From this screen, Dbr was identified as a candidate. Single esg+ cells are interspersed in young flies (Figure 1A). In aged flies, esg+ cells are clustered, enlarged in size, and aberrant in morphology (Figures 1B, 1E, and 1F; Biteau et al., 2008; Choi et al., 2008). Dbr was detected in different intestinal cell types (Figures S1A–S1C available online). When Dbr was depleted (Figure S1D), esg+ cells were often clustered, with aberrant morphology (Figures 1C–1E). Their cell size was increased, and many esg+ cells were equivalent in size to ECs (Figures 1C, 1D, and 1F). We also observed increased mitosis in these guts (Figure 1G). Because the observed defects were very similar to those observed in aged flies, we speculated that Dbr may be involved in one or more aging-related processes. In young flies, Dl protein (Delta) could only be detected in individual ISCs or one of two adjacent esg+ cells (Figure 1H). In aged flies, high levels of Dl were found in two or more adjacent esg+ cells of various sizes (Figure 1I). Moreover, in young flies, ee cells (by Pros) were rarely observed in esg+ cells. In aged flies, many Pros+ cells were also esg+ (Figures 1H and 1I). In dbr-depleted guts, we observed defects similar to those seen in aged flies (Figures 1J and 1K). The differentiation of ISC daughters is regulated by Notch signaling (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007). Notch signaling activation was determined by means of a Su(H)+GBE-lacZ reporter (Furriols and Bray, 2001). Notch signaling was activated in EBs in young flies (Figure 1L). In old flies, Notch signaling activation was widespread (Figure 1M). In control flies, Notch signaling was only activated in one of the two adjacent esg+ cells (Figure 1N). However, Notch signaling activation could be observed in many cells in esg+ clusters (Figures 1O and 1P). To further confirm these defects, we generated dbr MARCM clones (Lee and Luo, 2001). Two dbr EPI-001 Supplier were employed. The size of the ISC clones of both mutants was significantly increased compared with control clones, indicating increased ISC proliferation upon loss of dbr (Figures 1Q–1S and 2F). Consistent with the notion that the observed defects in dbr-depleted guts resulted from aberrant ISC proliferation/differentiation, these defects could be effectively suppressed by inhibition of epidermal growth factor receptor (EGFR) signaling (Figures S1E–S1H; Jiang et al., 2011). Together, these data demonstrate that Dbr is required for ISC proliferation/differentiation, thereby controlling midgut homeostasis. We further examined the mechanism(s) underlying Dbr-mediated ISC proliferation. A previous study showed that Dbr can define a Hh-responsive region in the wing disc by ubiquitination and lysosomal degradation of Ci (Dai et al., 2003). We first determined whether Ci is responsible for the defects observed in dbr mutants. Indeed, the defects caused by dbr knockdown could be suppressed by depletion of ci (Figures 1D and 2A–2C). Moreover, inhibition of ci alone could effectively inhibit ISC proliferation (Figures S2A–S2C). Furthermore, the size of the ISC clones of both dbr mutants was effectively suppressed by codepletion of ci (Figures 2D–2F), demonstrating that Ci is the cause of ISC proliferation in the absence of dbr. To further explore the role of Hh signaling in ISC behavior, we generated ptc mutant clones. Interestingly, the size of the ptc ISC clones was significantly increased compared with control clones (Figures 2G–2I). Moreover, in esg> ptc flies, the number of esg+ cells was substantially increased, forming clusters (Figures 2J–2L). These esg+ cells were morphologically aberrant, and many esg+ cells were increased in cell size (Figures 2J–2L), indicating that tissue homeostasis is lost upon Hh signaling activation. We also observed increased mitosis in ptc-depleted guts (Figure 2M). These phenotypes were very similar to those of aged flies and dbr mutants, suggesting that ectopic Hh signaling is responsible for ISC proliferation. Increased Dl was observed in two or more adjacent esg+ cells of various sizes. Many ee cells were also esg+ in ptc-depleted guts, indicating that homeostasis was lost due to increased ISC proliferation (Figure 2N). Widespread Notch signaling activation was observed in these guts (Figures 2O and 2P), indicating that aberrant progeny differentiation occurred. Lastly, ISC proliferation was increased upon ectopic Hh expression. These esg+ cells were aberrant in morphology. Meanwhile, some Pros+, esg+ cells were produced (Figures 2C and 2Q). Collectively, these data demonstrate that ectopic Hh signaling could result in ISC proliferation and misdifferentiation, leading to midgut homeostasis loss. Consistently, ISC proliferation was inhibited in esg > smo flies (Figures 2C and 2R). Similarly, the size of the smo ISC clones was significantly reduced (Figures 2S and 2T). Furthermore, we found that midgut regeneration under stress conditions was compromised by inhibition of Hh signaling (Figures S2D–S2G).