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  • GLPG0634 The aforementioned experiments demonstrated the ess

    2018-11-08

    The aforementioned experiments demonstrated the essential roles of Dbr/Hh signaling in midgut homeostasis. Next, we examined the source(s) of Hh involved in this process. We determined hh expression by using an hh-lacZ enhancer and Hh antibody. A high level of Hh expression was found in the anterior adult hindgut (Figures 3A and 3B; Takashima et al., 2008). In the posterior midgut, Hh expression was detected in the visceral muscles (VMs) and various intestinal GLPG0634 (Figures 3C–3H). Moreover, we found that Hh expression was significantly increased in aged and stressed flies (Figure S3A; Buchon et al., 2009). Next, we discriminated the source of functional Hh proteins via RNAi and various Gal4 drivers. However, ISC proliferation could still be observed after induction of hh (Figures 3I–3L; data not shown), indicating that Hh proteins from different sources play a redundant role in ISC proliferation. Consistently, Hh protein could be detected in the VMs and intestinal cells when ectopic hh expression was induced in the trachea and VMs, respectively (Figure S3B). These data indicate that Hh protein can be transported to the midgut epithelium from its various producing sources, consistent with our recent finding that Dpp (Decapentaplegic) can be transported from the trachea to the midgut epithelium (Li et al., 2013). Consistently, ISC proliferation was effectively suppressed in tub> hh flies and hh mutants (Figures 3L–3Q). Together, these data demonstrate that Hh from multiple sources is involved in midgut homeostasis. How does Hh signaling regulate midgut homeostasis? Damage by oxidative stress is one cause of age-related homeostasis loss. Previous studies have shown that JNK signaling increases cellular stress tolerance and contributes to tissue homeostasis loss in old and stressed guts (Biteau et al., 2008; Oh et al., 2005; Wang et al., 2003, 2005). Therefore, it is possible that Hh signaling may result in JNK signaling activation, causing midgut homeostasis loss. Indeed, we found that JNK signaling was dramatically elevated in aged flies (Figures 4A–4C). Moreover, Hh signaling activated by depletion of dbr or ptc substantially increased JNK signaling levels (Figures 4C–4F). Furthermore, JNK signaling activation in aged flies was greatly suppressed in esg> ci flies (Figures 4B and 4G). Importantly, midgut homeostasis loss caused by dbr knockdown was effectively suppressed by coinhibition of JNK signaling (Figures 4H–4O). These data argue that JNK signaling acts downstream of Hh signaling, contributing to tissue homeostasis loss. On the basis of our observations, we propose that Hh from multiple sources induces Hh signaling in ISCs to promote ISC proliferation/differentiation, causing tissue homeostasis loss. This detrimental effect of Hh signaling is safeguarded by Dbr, which maintains a proper level of Hh signaling in ISCs (Figure 4P). Our data suggest that JNK signaling acts downstream of Hh signaling in controlling tissue homeostasis. Our data demonstrate the essential role of Dbr in controlling tissue homeostasis. Stem cells are influenced by both intrinsic and extrinsic factors (Casali and Batlle, 2009; Radtke and Clevers, 2005; Xie and Spradling, 2000). Previous studies have demonstrated that fly ISCs receive various extrinsic signals from VMs and the midgut epithelium itself (Jiang et al., 2009, 2011; Lin et al., 2008). Our recent data also demonstrated that BMP from tracheal cells is an extrinsic signal required for midgut hemostasis (Li et al., 2013). The present study highlights Hh as another extrinsic signal that is essential for ISC physiology. Up to now, however, the intrinsic factors that control ISC behaviors have not been well studied. Here, we identified Dbr as an intrinsic factor. Proper control of the regenerative and self-renewal capacity of stem cells in the intestinal epithelium is of particular importance for long-lived organisms. Failure of this regulation in older individuals is reflected by the high incidence of intestinal adenomatous polyps (Radtke and Clevers, 2005). It has been reported that Shh levels are elevated in human gastric adenomas and intestinal metaplasias, suggesting that Hh overactivation may be involved in early gastric carcinomas (Parkin and Ingham, 2008). We found that the expression of Hh pathway components is increased in aging guts (Figure S4). Furthermore, although dbr expression increases as flies age, increased Hh signaling together with the activation of other signaling pathways, such as JAK/Stat and p38, may overcome the inhibitory effect of Dbr in aging flies, leading to midgut homeostasis loss (Figures S1A–S1C and S4).