Bidirectional Notch signaling regulates Drosophila intestinal stem cell multipotency

INTRODUCTION: In the Drosophila adult midgut, multipotent intestinal stem cells (ISCs) produce two types of daughter cells: nutrient-absorbing enterocytes (ECs) and secretory enteroendocrine (ee) cells. Notch signaling between ISCs and their daughters directs the proper specification of both of these cell types. Previous work suggests that ISCs expressing high levels of the Notch ligand Delta (Dl) strongly activate the Notch signaling pathway in their daughters and result in their differentiation into ECs. By contrast, ISCs that express low levels of Dl direct their daughters to become ee cells. However, in this unidirectional Notch signaling model, the mechanisms regulating differential Dl expression in ISCs are poorly understood. RATIONALE: During Drosophila pupal midgut development, pupal ISCs only make ee cells. Therefore, we examined how ee cells are made and evaluated the role of Notch signaling function during this developmental time window. On the basis of insights obtained from pupal development, we also asked whether similar mechanisms were used by ISCs in the adult midgut to generate ee cells. RESULTS: The ee cell fate marker Prospero (Pros) appeared in pupal ISCs at 44 hours after pupal formation (APF). From 44 to 96 hours APF, ISCs first divided asymmetrically, generating one ISC and one ee cell, followed by symmetric division of both ISCs and ee cells, resulting in a pair of ISCs and a pair of ee cells. During ISC asymmetric divisions, Pros was asymmetrically segregated to the basal daughter cell, a process that depended on the function of the Par complex. After ISC asymmetric division, the ee daughter cell expressed the Notch ligand Dl and activated the Notch signaling pathway in ISCs. Loss of Notch signaling in pupal ISCs induced all stem cells to differentiate into ee cells, whereas low-level activation of Notch signaling in pupal ISCs blocked ee cell formation. During ee symmetric divisions, Pros distribution was symmetric; however, cell polarity and Notch signaling remained asymmetric. Loss of Notch signaling between progeny of ee symmetric divisions disrupted expression of peptide hormones in ee cells, indicating a role for Notch signaling in proper ee specification.We also investigated the Notch pathway in adult ISCs and confirmed that postmitotic Notch signaling from ee daughter cells also regulates ISC multipotency. CONCLUSION: Consistent with previous work, high levels of Dl in ISCs activate high levels of Notch in the daughter cell, promoting EC differentiation. In contrast, after asymmetric localization of Pros, ISCs require a low Notch signal from their immediate ee cell daughters to maintain multipotency. Thus, Notch signaling is both bidirectional and context-dependent. Previous work also has suggested that ISCs remain basal during EC formation and that basal ISCs activate the Notch pathway in daughter cells. Our data show that ISCs are apically located during ee cell formation and that basal ee cells activate the Notch pathway in ISCs. Therefore, Notch signaling is always unidirectional in terms of polarity: Basal daughter cells express the Notch ligand Dl in order to activate the Notch signaling pathway in daughters after asymmetric ISC divisions. Our work provides further evidence that mechanisms regulating tissue homeostasis are more conserved between the Drosophila and mammalian intestine than previously thought. Inhibition of Notch signaling in the mouse intestine induces crypt base columnar stem cell loss and secretory cell hyperplasia, and ectopic Notch signaling promotes EC differentiation. Loss of Notch signaling in Drosophila ISCs also leads to stem cell loss and premature ee cell formation, whereas high Notch signaling promotes stem cell differentiation into ECs. Because Notch signaling also plays important roles in common lymphoid progenitors making B cells and T cells, and in airway basal cells making secretory cells and ciliated cells, it is tempting to speculate that bidirectional Notch signaling may regulate multipotency in these and other progenitors and stem cells.