© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 654-664 doi:10.1242/dev.114959

RESEARCH ARTICLE STEM CELLS AND REGENERATION Generation of enteroendocrine diversity in midgut stem cell lineages Ryan Beehler-Evans and Craig A. Micchelli*

ABSTRACT epithelium act broadly on endocrine, nervous and immune systems to The mediates long-range peptide hormone encode adaptive physiologic responses. Such peptides (also called signaling to broadcast changes in metabolic status to distant target neuropeptides) belong to distinct families, such as allatostatins, tissues via the circulatory system. In many animals, the diffuse diuretic hormones and tachykinins (Bendena et al., 1999; Gäde, 2004; endocrine system of the gut is the largest endocrine tissue, with the Nässel and Wegener, 2011; Van Loy et al., 2010). Knowledge differs full spectrum of endocrine cell subtypes not yet fully characterized. widely with respect to each peptide family member and their Here, we combine molecular mapping, lineage tracing and genetic experimentally defined roles within a given species. However, analysis in the adult fruit fly to gain new insight into the cellular and neuropeptide hormones have been implicated in processes such as molecular mechanisms governing diversity. growth control, muscle activity, feeding behavior, osmotic balance, Neuropeptide hormone distribution was used as a basis to generate a reproduction and epithelial secretion. The prodigious number of high-resolution cellular map of the diffuse endocrine system. Our enteroendocrine cells and their varied neuropeptide expression studies show that cell diversity is seen at two distinct levels: regional combine to make the gut the largest endocrine organ in the body. and local. We find that class I and class II enteroendocrine cells can And yet, the mechanisms underlying endocrine diversity in this be distinguished locally by combinatorial expression of secreted central endocrine tissue have not been fully understood. neuropeptide hormones. Cell lineage tracing studies demonstrate The invertebrate gastrointestinal (GI) tract, like its mammalian that class I and class II cells arise from a common stem cell lineage counterpart, contains a diffuse endocrine system providing a and that peptide profiles are a stable feature of enteroendocrine cell tractable experimental model to investigate the basis of enteroe- identity during homeostasis and following challenge with the enteric ndocrine diversity. This population of cells can be easily detected pathogen Pseudomonas entomophila. Genetic analysis shows that along the entire anterior-posterior (A/P) axis of the adult midgut Notch signaling controls the establishment of class II cells in the with the pan-enteroendocrine marker Prospero (Pros). Adult lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells are continuously maintained by stem cell enteroendocrine cells. Thus, one mechanism by which secretory cell divisions that occur in physiologically and functionally distinct diversity is achieved in the diffuse endocrine system is through cell- regions of the midgut (Micchelli and Perrimon, 2006; Ohlstein and cell signaling interactions within individual adult stem cell lineages. Spradling, 2006; Strand and Micchelli, 2011). Genomic analysis at the organ level has shown that a wide variety of neuropeptides and KEY WORDS: Drosophila, Diffuse endocrine system, Neuropeptide, their cognate receptors, many of which are conserved, are expressed Stem cell, Notch both within the gut and in the periphery (Marianes and Spradling, 2013; Buchon et al., 2013; Veenstra et al., 2008; Veenstra, 2009; INTRODUCTION Veenstra and Ida, 2014). It is also now possible to specifically On 16 January 1902, Ernest Starling observed a striking increase in isolate enteroendocrine cells and to obtain expression profiles at the pancreatic secretion when he prepared lysate from a segment of cellular level (Dutta et al., 2013). Moreover, experimental means to freshly excised intestinal mucosa rubbed with sand and weak HCl, challenge the barrier epithelium with pathogenic or nutritional filtered it through cotton wool and injected the extract into the insults are now available and combine readily with existing jugular vein of an anesthetized dog (Bayliss and Starling, 1902). molecular genetic tools (Vodovar et al., 2005; Lee and Micchelli, Documentation of this novel ‘reflex’ marked the inception of 2013; Piper et al., 2014). Thus, the GI tract of Drosophila provides as a field and implicated the as an unsurpassed experimental model to investigate aspects of diffuse a potent source of peptides capable of acting over a great distance via endocrine system biology. the circulatory system to profoundly alter physiology. We now know Studies of adult Drosophila enteroendocrine cells have focused that the gut epithelium is a rich source of secreted hormones that on the molecular events that determine whether a newly formed originate from specialized enteroendocrine cells and comprise a intestinal progenitor, called enteroblast, will adopt the secretory diffuse endocrine system distinct from the well-studied glandular (endocrine) or absorptive () cell fate (Micchelli and endocrine system (Engelstoft et al., 2013; Schonhoff et al., 2004). Gut Perrimon, 2006; Ohlstein and Spradling, 2006). The idea that Notch endocrine cells are embedded in the barrier epithelium and mediate signaling might control this decision was first suggested following luminal chemosensitivity (Furness et al., 2013; Reimann et al., 2012). the observation that high levels of a transcriptional reporter for In response to environmental stimuli, peptides secreted from the gut Notch could be detected in the enteroblast (Micchelli and Perrimon, 2006). Subsequent functional studies showed that the Notch ligand Washington University School of Medicine, Department of Developmental Biology, Delta can be detected at high levels in the neighboring intestinal 660 South Euclid Avenue, St Louis, MO 63110, USA. stem cell and that Delta is both necessary and sufficient *Author for correspondence ([email protected]) to distinguish the endocrine and enterocyte fate (Beebe et al., 2010; Ohlstein and Spradling, 2007). Together, these observations

Received 1 July 2014; Accepted 15 December 2014 led to a simple model in which stem cells signal to enteroblasts to DEVELOPMENT

654 RESEARCH ARTICLE Development (2015) 142, 654-664 doi:10.1242/dev.114959 influence cell fate: low levels of Notch transduction in the enteroblast promote the enteroendocrine differentiation and high levels of Notch transduction in the enteroblast promote the enterocyte differentiation. This asymmetry between stem cell and daughter cell is reinforced by segregation of Sara endosomes into the newly formed enteroblast (Montagne and Gonzalez-Gaitan, 2014). However, precisely when the specifying Notch signaling event occurs remains an open question (Perdigoto et al., 2011). Transcription factors of the Enhancer of split and achaete-scute complexes have both been shown to control endocrine differentiation (Bardin et al., 2010). More recently, Slit/Robo signaling has been implicated in enteroendocrine specification (Biteau and Jasper, 2014). Possible interactions between Notch and Robo signaling pathways have not yet been fully explored. The goal of the current study is to characterize the extent of cellular diversity in the diffuse endocrine system of adult Drosophila and to gain insight into the molecular mechanisms underlying this process. Here, we focus on the posterior midgut and use differential neuropeptide expression as a criterion to map enteroendocrine cell types in situ with great spatial resolution. We show that even immediately adjacent endocrine cells along the midgut express different combinations of neuropeptides, dividing the diffuse endocrine system into class I and class II endocrine cells. Directed cell lineage-tracing studies show that, once an enteroendocrine cell subtype is established, it is stably maintained in the adult, even following environmental challenge. Thus, diversity of endocrine cells is seen at two levels: regional and local. We demonstrate that individual gut stem cells generate pairs of class I and class II enteroendocrine cells displaying distinct neuropeptide profiles. Moreover, the precise combination of neuropeptide expression depends on stem cell position within the midgut. Finally, our analysis shows that Notch signaling is initially required to generate class II enteroendocrine cell diversity in posterior midgut stem cell lineages, but is dispensable for both underlying regional identity and neuropeptide expression in extant endocrine cells. We conclude that local endocrine diversity is generated in the diffuse endocrine system during adult homeostasis through cell-cell signaling interactions within individual stem cell lineages.

RESULTS Neuropeptide expression distinguishes adult enteroendocrine cells in the posterior midgut The adult Drosophila midgut epithelium is a rich source of secretory neuropeptide hormones, showing elevated levels of Allatostatin A (AstA), Allatostatin B (AstB), Allatostatin C (AstC), Neuropeptide F (Npf), Diuretic hormone 31 (DH31) Fig. 1. Diversity of adult enteroendocrine cells is both regional and local. and Tachykinin (Tk) (Veenstra et al., 2008; Reiher et al., 2011; (A) Regional differences in peptide hormone expression distinguish segments Veenstra and Ida, 2014). Neuropeptide expression subdivides the of the adult posterior midgut (shading). (B) Within each region, local gut into broad regions along the A/P axis, which are evident with differences in peptide hormones are detected among endocrine cell pairs. regards to both anatomical and molecular markers (supplementary Class I and II enteroendocrine cells are distinguished by peptide antisera and Gal4 lines. Yellow indicates Notch-dependent cell fates. DH, DH31. material Fig. S1A-N; Table S1) (Veenstra et al., 2008; Veenstra and + (C-H) Peptides are differentially detected in Pros cell pairs. (C) Anti-AstA, red; Ida, 2014). For example, the posterior midgut is divided into two anti-Pros, red. (D) Anti-AstB, red; anti-Pros, green. (E) Anti-AstC, red; anti- primary neuropeptide-expressing regions that we refer to as region Pros, green. (F) Anti-DH31, red; anti-Pros, green. (G) Anti-Npf, red; anti-Pros, 1 and region 2, respectively (Fig. 1A; supplementary material green. (H) Anti-Tk, red; anti-Pros, green. (I) Pairs of endocrine cells express Fig. S1A,B, Fig. S2A-F; Table S1). A panel of transgenic different peptides. Anti-AstA, red; anti-Tk, green. (J) At the transition between molecular markers have been used to subdivide the posterior region 1 and 2, AstA increases posteriorly, whereas AstB decreases midgut into regions (Marianes and Spradling, 2013; Buchon et al., posteriorly. Anti-AstA and anti-AstB, white; Tk>GFP, green. Scale bars: 10 µm. 2013). However, careful comparison of relevant marker strains with neuropeptide antisera revealed segmental boundaries that peptide-rich posterior midgut, we use the ‘region 1’ and ‘region 2’ were distinct (supplementary material Fig. S2A-F). Therefore, designations to refer directly to native domains of peptide for the purpose of this study, which focuses primarily on the expression in the adult midgut. DEVELOPMENT

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Adult stem cells maintain the differentiated epithelial lining of anterior and middle regions of the adult midgut (Fig. 1G; the midgut (Micchelli and Perrimon, 2006; Ohlstein and supplementary material Fig. S1; Table S1). Thus, precise Spradling, 2006). This monolayer consists primarily of large combinations of secretory neuropeptides can be used to absorptive enterocyes, but also includes ∼1000 (∼10%) secretory distinguish each cell within an enteroendocrine pair 5 days after enteroendocrine cells that can be identified by the pan-endocrine eclosion. marker Pros. However, it has been unclear whether neuropeptide expression in the midgut is restricted only to endocrine cells, or Neuropeptide expression is a fixed characteristic of the extent to which individual cells might express specific enteroendocrine cell subtypes combinations of particular neuropeptides. To determine whether Neuropeptides actively modulate organismal physiology. It is therefore secretory neuropeptides are expressed in Pros+ cells, we possible that established cellular profiles of peptide expression conducted a series of double-labeling experiments on five-day- associated with a class of enteroendocrine cells can change during old adult midguts. Our studies show that distinct subsets of Pros+ adulthood. Alternatively, peptide expression might be a fixed cells also stain positive for AstA, AstB, AstC, DH31, Npf and characteristic of endocrine cell subtypes. To test these possibilities, Tk when assayed in a pairwise manner (Fig. 1C-H). Initial we conducted directed cell lineage-tracing experiments using specific characterization indicated that, with the exception of AstC in neuropeptide Gal4 driver lines (Fig. 2). Class I enteroendocrine cells in region 1, only one of the two adjacent Pros+ cells stained positive region 2 of the posterior midgut were genetically labeled by crossing for the peptide assayed. This impression was confirmed by scoring the percentage of Pros+ cells within a given region that also expressed a particular neuropeptide (Table 1). Values ranged from 48 to 52%, or approximately half of the Pros+ cells. Neuropeptide expression was specific to endocrine cells, with no obvious expression detected above background levels elsewhere in the gut epithelium. These results were independently confirmed using specific neuropeptide Gal4 driver lines, which we found to be expressed in the adult midgut (Materials and Methods; supplementary material Fig. S1F,J,N and Fig. S3; Table S1). Thus, neuropeptide expression in the gut is limited to endocrine cells, and in all but one case, labels half of the Pros+ cells in a particular midgut region. Adjacent Pros+ enteroendocrine cells can be unambiguously distinguished by unique neuropeptide expression profiles (Fig. 1B,I; supplementary material Fig. S1A-N and Fig. S3A-N; Table S1). A comprehensive series of double- and triple-staining studies, employing both antibodies and Gal4 lines, was performed. This analysis showed that in region 1, for example, class I enteroendocrine cells are AstB+,AstC+, whereas class II enteroendocrine cells are AstC+ (supplementary material Fig. S3E,F). In region 2, class I enteroendocrine cells are AstA+,AstC+ (supplementary material Fig. S3C,D,G,H), whereas class II enteroendocrine cells are DH31+, Tk+ (supplementary material Fig. S3I,J,M,N). To determine which fraction of Pros+ cells express either class I or class II neuropeptide markers we simultaneously labeled AstC+,Tk+ and Pros+ cells (Table 1). Using combinations of either antisera or Gal4 lines revealed that the vast majority (>95%) of Pros+ cells also express neuropeptides. A transition zone between region 1 and region 2 was identified, where neuropeptide protein is detected in a graded manner within class I endocrine cells (Fig. 1J; supplementary material Table S1). We note that pairs of class I and class II enteroendocrine cells were also detected in the

Table 1. Distribution of neuropeptides in Prospero+ cells Neuropeptide+/ Antigen Neuropeptide+ Pros+ Pros+ ratio (%) Region n (guts) AstA 106 206 51 2 6 AstB 42 88 48 1 3 Fig. 2. Peptide expression stably distinguishes class I and II AstC 261 269 97 1 6 enteroendocrine cells. (A-H) Directed lineage tracing of class I and II DH31 196 383 51 2 6 enteroendocrine cells using actin5C>lacZ; UAS-flp. Anti-β-Gal, red; anti-AstA/ Npf 245 500 49 Anterior+CC 6 anti-DH31, green. (A,C,E,G) AstA-Gal4. (B,D,F,H) Tk-Gal4. (A,B) Gal4 lines Tk 252 525 48 2 6 are not detected in newly eclosed adult midguts. (C,D) 4 days following AstC/Tk 749 792 95 1+2 6 eclosion Gal4 drivers mark endocrine subtypes. Marked cells are distinguished GFP* 1487 1557 96 1+2 8 from their neighbors with neuropeptide antisera. (E,F) 10 days following

*AstC-Gal4/Tk-Gal4×UAS-GFP; CC, Copper cell region. eclosion. (G,H) Challenge with Pe. Scale bar: 10 µm. DEVELOPMENT

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Table 2. Directed cell lineage tracing of enteroendocrine cell subtypes AstA>Trace Timepoint (days) DH31+β-Gal+ β-Gal+ DH31+β-Gal+/β-Gal+ ratio (%) n (guts) 0000 5 40540 4 10 1 197 <1 7 8* 0 161 0 6 Tk>Trace Timepoint (days) AstA+β-Gal+ β-Gal+ AstA+β-Gal+/β-Gal+ ratio (%) n (guts) 0000 6 4 0 146 0 7 10 0 244 0 8 8* 1 161 <1 7 *24hPe challenge. the AstA-Gal4 to the actin5C FRT stop FRT lacZnuc; UAS-flp lineage Characterization of wild-type lineages was also performed in tracer (AstA>Trace; Fig. 2A,C,E,G). The AstA-Gal4 driver line was region 1 of the posterior midgut, yielding similar results. However, first detected in the adult midgut 4 days following eclosion (Fig. 2A,C). analysis of the region 1 was limited by the lack of markers to Ten days following eclosion, midguts were stained to identify DH31+ simultaneously distinguish class I and class II enteroendocrine cells, a marker of class II enteroendocrine cells in region 2 of the cells (e.g. Fig. 1B and Fig. 3F,G; supplementary material posterior midgut. We observed that cells labeled by AstA-Gal4 were Table S1). Thus, we scored the fraction of Pros+ cells that were DH31−, although DH31 staining was readily detected in adjacent also AstB+ (Fig. 3H,I; Table 4). We predicted that half of the Pros+ endocrine cells (Fig. 2E). In a complementary experiment, we labeled enteroendocrine cells present in each clone would be AstB+.We class II enteroendocrine cells in region 2 using Tk-Gal4 (Fig. 2B,D,F, found that marked lineages recovered in region 1 contained H). The Tk-Gal4 driver line was also first detected in the adult midgut between0and3Pros+ cells. However, among the clones that 4 days following eclosion (Fig. 2B,D). Ten days following eclosion, contained two Pros+ cells, 48% fit this criterion (Table 4). In we stained for AstA+, a neuropeptide that marks class I enteroendocrine summary, nearly half of the wild-type clones observed in region 1 cells in region 2. We observed that cells labeled by Tk-Gal4 were were consistent with a model in which class I and II cells arise AstA−, although AstA+ cells were readily detected in adjacent from a common lineage, as shown for region 2. It is likely that the endocrine cells (Fig. 2F). To further document these observations we method of clone scoring used for analysis in region 1 enriched for counted the number of lacZ+, DH31+ and lacZ+,AstA+ cells present in a set of early clones containing less differentiated cells (i.e. Pros+ adult midguts at 0, 4 and 10 days after eclosion, and again 24 h only). By contrast, the analysis in region 2 was biased toward late following challenge with the Gram-negative enteric pathogen clones identified by the presence of highly differentiated Pseudomonas entomophila (Pe) (Fig. 2G,H; Table 2). This analysis endocrine cells (i.e. AstA+,Tk+). Taken together, analysis of shows that AstA-Gal4 and Tk-Gal4 driver lines mark endocrine cell wild-type cell lineages supports a general model in which class I subtypes that remain DH31− or AstA− during adult homeostasis, as and class II endocrine cell pairs of the posterior midgut arise from well as following pathogenic challenge. Taken together, these a common stem cell lineage. experiments suggest that the class I and class II neuropeptide profiles remain stable in enteroendocrine cells during adult gastrointestinal Class II enteroendocrine cells arise from a Su(H)-Gal4 homeostasis as well as following pathogenic challenge. Thus, expressing progenitor neuropeptide expression provides a reliable means of distinguishing The choice of cell fate between absorptive and secretory distinct enteroendocrine cell subtypes in the adult midgut. endocrine cells in the midgut is controlled by the Notch signaling pathway (Bardin et al., 2010; Beebe et al., 2010; Micchelli and Class I and class II enteroendocrine cells are lineally related Perrimon, 2006; Ohlstein and Spradling, 2006, 2007; Perdigoto The presence of enteroendocrine cells with distinct neuropeptide et al., 2011). Activation of Notch signaling in stem cell daughters, expression profiles raised the question whether such cells might called enteroblasts, promotes enterocyte formation, whereas reduced arise from a common cell lineage or whether they are lineally Notch signaling leads to the production of ectopic enteroendocrine distinct (Fig. 3A). To distinguish these possibilities, we cells. This model predicts that directed cell lineage-tracing of conducted cell lineage-tracing studies. Wild-type lineages were progenitors transducing high levels of Notch signaling should label labeled using the MARCM system (Lee and Luo, 1999) and only enterocytes. We tested this prediction using a pulse/chase stained to identify enteroendocrine subtypes. We focused first on experiment and the conditional Su(H)-Gal4, tub-Gal80TS strain to region 2 of the posterior midgut, where simultaneous detection of label a fraction of adult cell lineages with actin5C FRT stop FRT class I and II enteroendocrine cells was possible. Here, marked lacZnuc; UAS-flp under both baseline conditions and following lineages were found to span both class I and II endocrine cell environmental challenge (Table 5; Fig. 4A-D). As predicted, subtypes (Fig. 3B-E). To further characterize the distribution of enterocytes were readily labeled by this procedure when cell endocrine cell subtypes, the number of AstA+ and Tk+ cells per marking was initiated, following a shift to the non-permissive clone was scored for each marked lineage recovered (Table 3). temperature (Fig. 4A-D). However, quite unexpectedly, we also The total number of AstA+ and Tk+ cells per clone ranged from 0 recovered labeled Tk+ class II endocrine cells at a frequency of to 4. In every case in which a marked lineage contained two or 0.65% under baseline conditions (Table 5; Fig. 4A). Similarly, we more endocrine cells, we scored the presence of both AstA+ and observed labeled DH31+ cells, a second marker correlated with class Tk+ enteroendocrine cells. We conclude that in region 2 of the II endocrine fate, following environmental challenge (Table 5; posterior midgut, class I and II enteroendocrine cell pairs arise Fig. 4C). By contrast, Tk+ class II cells were never labeled in + from a common cell lineage. unshifted control experiments; nor were AstA class I cells labeled at DEVELOPMENT

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Table 3. Analysis of neuropeptides in region 2 cell lineages WT 0 AstA+ 1AstA+ 2AstA+ ≥3AstA+ 0Tk+ 31 8 0 0 1Tk+ 51010 2Tk+ 0210 ≥3Tk+ 0000 N55e11 0AstA+ 1AstA+ 2AstA+ ≥3AstA+ 0Tk+ 89 0 0 29 1Tk+ 0000 2Tk+ 0000 ≥3Tk+ 0000

signaling in the generation of regional diversity among midgut endocrine cell subtypes.

Class II enteroendocrine cells are established by Notch/ Delta signaling Notch loss in the intestinal stem cell lineage leads to the production of ectopic Pros+ cells, suggesting the formation of enteroendocrine tumors (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Yet, the full extent of endocrine cell differentiation in these tumors is unclear. Therefore, we first confirmed and extended previous studies by generating null Notch clones and monitoring the endocrine fate using an expanded panel of endocrine cell markers, including the pan-endocrine marker Pros; the ELKS family protein Bruchpilot (Brp), which supports calcium-dependent secretory vesicle release; and cell morphology. In addition, we examined the levels of Peptidylglycine α-hydroxylating monoxygenase (Phm). Phm is an enzyme that catalyzes the rate-limiting step in C-terminal α-amidation, a necessary step for the production of active neurosecretory peptides from pro-forms of the peptides. We found that Notch loss led to ectopic cells displaying the characteristic morphology of mature enteroendocrine cells with polarized cellular processes projecting toward the gut lumen (Fig. 5A). In addition, these cells were Pros+, Brp+ and Phm+ (Fig. 5B-D). Thus, we conclude that the ectopic cells that arise in Notch mutant lineages exhibit multiple features of well-differentiated endocrine tumors. By contrast, Notch loss was found to differentially affect the distribution of specific secretory neuropeptides. We examined distinguishing markers for both class I and class II enteroendocrine cells in region 2 of the posterior midgut (Fig. 5E-T). Notch clones in region 2 led to loss of both DH31+ and Tk+, whereas AstA+ cells remained present in the lineage (Fig. 5E-L). We quantified this phenotype and found that, in every case in which two or more endocrine cells were present, clones retained AstA+ cells but lost Fig. 3. Class I and II enteroendocrine cells arise from a common lineage. Tk+ cells (Table 3). Similar results were observed using additional (A) Wild-type class I (pink) and class II (red) endocrine cells are present in class I and class II markers in the anterior midgut, copper cell region pairs distributed among enterocytes (gray). Alternate lineage models are and region 1 of the posterior midgut (Fig. 5M-T; supplementary distinguished by cell-labeling studies (green). (B-I) Wild-type MARCM lineages material Fig. S4; Table S4). Importantly, these findings were 5 days after induction, dashed line. (B-E) Lineages spanning class I and II entoendocrine cells, region 2. Anti-AstA, red; anti-Tk, green. Distinct endocrine subtypes arise from a common lineage. (F-I) Marked lineages, region 1. Anti- Table 4. Analysis of neuropeptides in region 1 cell lineages Pros, white. (G) Anti-AstC, red. (I) Anti-AstB, red. Scale bar: 10 µm. WT 0Pros+ 1Pros+ 2Pros+ 3Pros+ 0AstB+ 92 15 27 2 1AstB+ – 9282 the non-permissive experimental temperature (Table 5). This 2AstB+ ––33 suggested that Notch signaling might be active specifically in the 3AstB+ ––– 0 class II enteroendocrine cell lineage. Consistently, Notch pathway + + + AstB Pros / reporters were also detectable in Tk cells (Fig. 4E-H). From this, we Genotype AstB+Pros+ Pros+ Pros+ ratio (%) n (guts) conclude that DH31+ and Tk+ class II enteroendocrine cells arise from a Su(H)-Gal4-expressing progenitor. These findings prompted WT 51 161 32 18 N55e11 149 249 60 7 a series of experiments to directly test the function of Notch DEVELOPMENT

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Table 5. Analysis of Su(H)>Trace Peptide+β-Gal+/ ‘Chase’ n Peptide Peptide+β-Gal+ β-Gal+ β-Gal+ ratio (%) (days) (guts) Tk 0 9 0 10* 6 ‡ Tk 11 1690 0.65 10-12 5 ‡ AstA 0 1141 0 10-12 5 Pros 5 731 0.68 10§ 5 DH31 8 1214 0.71 14§ 10 DH31 19 2693 0.71 15¶ 10 ‡ *Unshifted, unchallenged; shifted, unchallenged; § shifted, challenged (heat stress); ¶shifted, challenged (Pe). independently confirmed using conditional Notch knockdown (supplementary material Fig. S5A-J). We note that, whereas Notch loss affected local neuropeptide profiles among endocrine pairs, it was not associated with changes in underlying regional identity in neuropeptide expression (supplementary material Fig. S5K). Thus, Notch signaling is necessary to specify the class II endocrine cell subtype in the adult midgut, without affecting neuropeptide processing enzymes or the cellular machinery implicated in secretory vesicle release. The Notch pathway can be activated by Delta and Serrate ligands. As in the case of wild type, marked lineages lacking Serrate produced both AstA+ and Tk+ endocrine cells (Fig. 6A,B). However, loss of Delta was necessary for the production of Tk+ enteroendocrine cells (Fig. 6C-F). We note that Delta clones exhibited greater heterogeneity in the progenitor marker esg-lacZ than Notch, which correlated with heterogeneity of the neuropeptide phenotype (Fig. 6G,H; supplementary material Fig. S6). Thus, Delta, but not Serrate, is necessary for establishing class II enteroendocrine cells during adult midgut homeostasis.

Notch function is dispensable for endocrine cell maintenance Notch might function only in the establishment of endocrine diversity. Alternatively, Notch might function in both the establishment and maintenance of endocrine diversity. To distinguish these possibilities, we pursued a conditional, cell type- specific genetic loss-of-function approach. Conditional Notch Fig. 4. Class II endocrine cells arise from a Su(H)-Gal4 progenitor. knockdown was achieved in extant class II enteroendocrine cells (A-D) Conditional lineage tracing of Su(H)-Gal4TS cells using actin5C>lacZ; using the UAS-GFP, tub-Gal80TS; Tk-Gal4 strain. However, despite UAS-flp, 12 days after induction. (A,B) Cellular products of Su(H)-Gal4TS strong Notch knockdown with this construct (e.g. supplementary labeling, red/white; anti-Tk, green. Arrows indicate Tk+ cells derived from Su TS + material Fig. S5), no change in either DH31+ or AstA+ cells was (H)-Gal4 cells. Asterisks show unlabeled Tk cells. (B) Single channel. (C,D) Cellular products of Su(H)-Gal4TS, 7 days following exposure to Pe, red/ detected (supplementary material Fig. S7A-D). This suggests that white; anti-DH31, green. Arrows indicate DH31+ cells derived from Su(H)- Notch signaling is dispensable for maintenance of neuropeptide Gal4TS cells. Asterisks show unlabeled DH31+ cells. (D) Single channel. expression in enteroendocrine cells. (E-H) Markers of Notch signaling in Tk+ cells, red. Arrows indicate Tk+ cells. Asterisks show unlabeled Tk+ cells. (E,F) Su(H)GBE-lacZ reporter. Note + Notch activation is sufficient to establish class II lower levels of the reporter in the Tk cells. (E) esg>GFP, green; anti-Tk, red. enteroendocrine cells (F) Su(H)GBE-lacZ, white. (G,H) Dl-lacZ labeling, red/white; anti-Tk, green. We tested whether Notch signaling is also sufficient to establish (H) Single channel. Scale bars: 10 µm. local differences in endocrine cell subtype. In a first test, we examined the consequences of global Notch activation on material Fig. S8C,D; Table 3). This observation is consistent with endocrine cell identity by monitoring DH31+ cells at 6 and 24 h the idea that Notch activation is sufficient to promote class II following induction of an hs-Notchintra transgene (supplementary endocrine cell fate. The low frequency phenotype is predicted material Fig. S8). To distinguish newly formed cells from because endocrine progenitors are an order of magnitude rarer previously differentiated cells, we performed the experiment in than epithelial progenitors and because, under baseline an esg>GFP background. As previously reported, we readily conditions, proliferation of all progenitors is infrequent. We observed that Notch pathway activation promoted differentiation reasoned that, if progenitor pools are first increased, using genetic of esg>GFP+ into large enterocytes 24 h following induction means, and then Notch is ectopically activated, the phenotype (supplementary material Fig. S8A,C). We also observed a low should be detected with greater frequency. Therefore, in a second frequency of DH31+ doublets under these conditions, which are test of Notch sufficiency, conditional Presenillin knockdown with TS not typically seen in wild-type cell lineages (supplementary esg-Gal4; UAS-GFP, tub-Gal80 was followed by induction of DEVELOPMENT

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Fig. 5. Notch is necessary to establish class II endocrine cells. (A-T) Mosaic analysis of Notch using MARCM; representative clones are highlighted by dotted lines. (A-D) General endocrine markers, 7 days after induction. (A) Anti-GFP, green; phalloidin, red, staining F-actin (muscle). (B) Anti-Pros, red. (C) Anti-Brp, red. (D) Anti-Phm, red. (E-T) Distribution of neuropeptides in Notch clones, 5 days after induction. Clones on left in paired micrographs, green. Asterisks, internal controls. (E-L) Region 2. (F,H) Anti-AstA, red; anti-Tk, green. Endocrine cell morphology is evident in cross-section. (I-M,O,Q,S) Anti-Pros, red. (J,L) Anti-DH31, green. Note the absence of DH31+ cells within the clone. (M-T) Region 1. (N,P) Anti-AstC, red. (R,T) Anti-AstB, red. Scale bars: 10 µm. the hs-Notchintra transgene. Under these conditions we observed a adult midgut homeostasis. However, once a class I endocrine cell rapid, robust and significant increase in the number of Pros+, is established, ectopic Notch activation is insufficient to DH31+ cells (Fig. 7A-F). Finally, we tested whether Notch reprogram subtype identity. activation is sufficient to reprogram fully differentiated enteroendocrine cell fate from class I to class II. However, DISCUSSION conditional activation of Notchintra in AstA+ cells was insufficient The barrier epithelium of the gut is a unique biological interface. It to induce DH31 peptide in these cells (Fig. 7G,H). Altogether, is here that a complex stream of continuously changing luminal these results suggest that Notch signaling is both necessary and stimuli are presented to the organism and must be converted into sufficient to specify the class II endocrine cell subtype during appropriate physiologic and neuromodulatory states. The cells of DEVELOPMENT

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indicates that >95% of Pros+ cells express one or more distinct neuropeptides. We predict that the remaining ∼5% of Pros+ cells correspond either to newly formed endocrine cells in the process of differentiating, dividing progenitors (see Biteau and Jasper, 2014; Zielke et al., 2014) or differentiated endocrine cells expressing peptides other than those tested. The mapping data presented here confirm and extend a rapidly growing body of work, which has provided essential insight into the distribution of regulatory neuropeptides in Drosophila (Buchon et al., 2013; Chintapalli et al., 2007; Marianes and Spradling, 2013; Reiher et al., 2011; Veenstra et al., 2008; Veenstra and Ida, 2014). Overall, these reports are in general agreement, despite significant differences in approach, methodology and scope. Our systematic, combinatorial mapping strategy provides high cellular and temporal resolution of neuropeptide distribution in vivo under baseline conditions. A pairwise comparison of available antisera with Gal4 driver lines suggests that patterns of neuropeptide expression and protein distribution are similar, if not identical, in the adult midgut. Importantly, these validated reagents provide powerful and specific new tools for manipulating the endocrine system, as we demonstrate. Finally, directed cell lineage analysis shows that neuropeptide profiles are stable features of endocrine cells during both normal homeostasis and following challenge with the enteric Gram-negative pathogen Pe. In mammals, the classical view of the diffuse endocrine system is the ‘one cell-one hormone’ dogma (Engelstoft et al., 2013; Schonhoff et al., 2004). However, recent studies in mouse and human have challenged this perspective (Egerod et al., 2012; Habib et al., 2012; Sei et al., 2011). It is now clear that the mammalian GI tract exhibits not only regional diversity but also local diversity in the combinatorial expression of neuropeptides in single endocrine cells of the diffuse endocrine system. For example, in the murine intestine, endocrine cells in the crypt express multiple endocrine hormones, but endocrine cells on the villus do not (Sei et al., 2011). Interestingly, in mammals, peptides that are coexpressed also appear to have overlapping or coordinated functions. Thus, broad coexpression of secretory neuropeptides is a conserved feature of the diffuse endocrine system among metazoans. Fig. 6. Delta is necessary to establish class II endocrine cells. (A-H) Analysis of Delta and Serrate lineages using MARCM, dotted line. Class I and class II endocrine subtypes arise from a common (A,B) Serrate clone, 5 days after induction. (B) Anti-AstA, red; anti-Tk, green. stem cell lineage (C,D) Delta clone, 5 days after induction. (D) Anti-AstA, red; anti-Tk, green. The diffuse endocrine system differs from the glandular endocrine + Asterisk shows Tk cell outside clone. (E,F) Delta clone, 10 days after system in that it must be continuously renewed. The combinatorial induction. (F) Anti-AstA, red. (G,H) Delta clone, 10 days after induction. differences among endocrine neuropeptides described here provide (H) Anti-AstA, red; esg-lacZ, white. Scale bars: 10 µm. an experimental basis to address how diversity is maintained in the diffuse endocrine system. We distinguish two possible models and the diffuse endocrine system represent an early, if not the primary, directly demonstrate that endodermal stem cell lineages give rise to level at which these changes are sensed and broadcast. We have used both class I and class II endocrine cells from a single lineage. Thus, Drosophila as a model system to address the molecular mechanisms local diversity within the diffuse endocrine system is controlled by by which cellular diversity in the diffuse endocrine system is individual stem cell lineages situated at distinct positions along the homeostatically maintained. length of the GI tract. Developmental studies have characterized the stepwise process of endocrine cell development (Micchelli et al., Organization of the adult enteroendocrine system 2011; Takashima et al., 2011). Cell divisions occurring during late Endocrine cells can be characterized by the neuropeptides they pupal stages initially establish pairs of gut enteroendocrine cells. secrete. From this perspective, the adult diffuse endocrine system Moreover, recent studies have implicated the gut neurosecretory exhibits both regional and local organization. We show that peptide Bursicon as a key peptide in the complex behavioral process neuropeptide expression in the adult midgut is restricted to cells of pupal eclosion (Scopelliti et al., 2014). The fact that cellular positive for Pros, a pan-endocrine marker defining the extent of the diversity in the diffuse endocrine system is actively maintained by diffuse endocrine system. Among Pros+ cells, distinct segmental adult stem cell lineages suggests that endocrine cells have broad regions of neuropeptide expression along the A/P axis are evident. functional significance in maintaining many aspects of metabolic Within each region, local differences in combinatorial neuropeptide homeostasis throughout the course of adulthood. These crucial distribution distinguish class I and II endocrine cells. Our analysis functions remain to be fully characterized. DEVELOPMENT

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Fig. 7. Notch activation is sufficient to establish class II enteroendocrine cells. (A-F) Analysis of Notch pathway activation on endocrine cell fate, 8 days following conditional Presenilin knockdown using esgTS. Adults were shifted to the non-permissive temperature for 7 days to induce Presenilin knockdown and then stained against DH31+ cells 24 h later, in the presence or absence of global Notch pathway activation. (A,C,E) Anti-Pros, red. (A-D) Anti-DH31, green. (A,B) Presenilin knockdown leads to endocrine hyperplasia and a reduction in DH31+ cells. (C,D) Presenilin knockdown leads to endocrine hyperplasia and a rapid increase in DH31+ cells following global Notch activation. Inset, DH31+ cells exhibit endocrine morphology. (E) Notch activation is sufficient to promote differentiation of esg+ cells to large enterocytes, green. (F) Quantitation. The average number of DH31+ cells per gut scored for each genotype (n=7-12 guts per genotype, error bars represent s.e.m.; ***P<0.0001). (G-H) Analysis of Notch pathway activation in fully differentiated class I endocrine cells using AstA-Gal4TS. Adults were shifted to the non-permissive temperature for 7 days. AstA>GFP, green; Anti-DH31, red. (G) Controls shifted to non-permissive temperature show conditional expression of GPF. (H) Notch overexpression in AstA+ cells does not cause expression of the class II marker DH31. (I) Schematic cell lineage diagram. Undifferentiated stem cell daughters (enteroblasts) give rise to either the secretory (enteroendocrine) or absorptive (enterocyte) cell fate. Branch points indicate Notch-dependent cell fate decisions. Progenitors, green; differentiated cells, pink, red and blue; Notch-dependent cell fates, yellow. ISC, intestinal stem cell; EB, enteroblast. Scale bars: 20 µm.

progenitors were found to give rise to class II, but not class I, endocrine cells, under a wide variety of experimental conditions that included baseline homeostasis and different environmental challenges, suggesting a key role for the Notch signaling pathway in generating enteroendocrine diversity. We note that this finding differs significantly from another report, which concluded that endocrine cells do not arise from a Su(H)Gal4+ progenitor (Biteau and Jasper, 2014). For example, in one experiment, we detected five Pros+ cells among a total of 731 labeled cells (10 days following induction, five posterior midguts), whereas Biteau and Jasper report no Pros+ cells among 418 (4 days following induction, eight guts). This disparity is therefore most likely explicable by the short chase period used and the relative rarity of endocrine progenitors in the gut. Our subsequent functional characterization clearly showed that Notch signaling is both necessary and sufficient to control cell diversity in the diffuse endocrine system. Although Notch loss leads to well-differentiated endocrine tumors, they exhibit specific changes in the type of endocrine cells produced. By contrast, Notch function was dispensable in controlling underlying regional identity along the length of the gut. Gaining insight into the process underlying regional specification represents an interesting line of future investigation. Although our analysis focused on the posterior midgut, where peptide diversity is greatest, our studies also indicate that similar requirements for Notch in class II endocrine cells exist along the entire length of the GI tract. Finally, targeted manipulation of Notch signaling in differentiated endocrine cells failed to alter endocrine identity, consistent with a model in which Notch signaling functions specifically in endocrine progenitors. Thus, Notch signaling plays at least two separable roles in controlling cell fate within the gut stem cell lineages: a previously known requirement in specifying absorptive enterocytes, and the newly described role in controlling the diversity of secretory enteroendocrine cells along the length of the adult midgut demonstrated here (Fig. 7I, yellow). Non-autonomous consequences of endocrine tumors are implicit by the very nature of the peptides these cells secrete. Indeed, tumors are neuroendocrine malignancies that secret Controlling cell type in the endocrine lineage hormones, which cause debilitating symptoms in patients. In Simultaneous detection of class I and II endocrine cells in marked addition, loss of endocrine cell subtypes has been associated with endodermal lineages suggested that endocrine cell fate is controlled metabolic disruptions, including lipid absorption and glucose by signaling events within individual stem cell lineages. Su(H)-Gal4 homeostasis (Mellitzer et al., 2010). Our study suggests that these DEVELOPMENT

662 RESEARCH ARTICLE Development (2015) 142, 654-664 doi:10.1242/dev.114959 effects might result from either the loss of specific peptides or supplemented with yeast paste at 25°C. Newly eclosed females of the overabundance of others in the diffuse endocrine system. Thus, appropriate genotype were aged 3-5 days prior to clone induction. Clones further insight into how endocrine peptides are produced and how were induced by placing vials in a 37°C water bath for 30-40 min. Flies were secretion is controlled might have important consequences for heat-shocked 2-3 times within a 24 h period. understanding tumor pathophysiology and for developing targeted treatments for metabolic disease. Pe infection Flies were infected ad libitum with Pe. Infected flies were fed on food supplemented with 0.5 ml of Pe at OD5-OD20 in 5% sucrose. Mock-infected MATERIALS AND METHODS flies were placed on food supplemented with 0.5 ml 5% sucrose. Flies were Fly stocks infected with Pe for 24 h and subsequently maintained at 29°C throughout 1118 The following fly stocks were used: from Bloomington Stock Center: w the course of the experiment. (#39352; wild-type control); Canton-S (#1; wild-type control); w1118; GMR65D06-Gal4 (#39352; AstA-Gal4, this study); w1118;GMR61H08- 1118 Conditional manipulation of Notch pathway Gal4 (#39283; AstC-Gal4, this study); w ;GMR57F07-Gal4(#46389; To conditionally target progenitor cells or differentiated endocrine cell DH31-Gal4, this study); w1118;GMR61H07-Gal4(#39282; called Tk-Gal4, 1118 1118 populations in the adult midgut, esg-Gal4, Tk-Gal4 or AstA-Gal4 was first this study); w ; GMR50A12-Gal4/TM3 (#47618); w ;GMR42C06- TS RNAi 1118 10419 506 combined with tub-Gal80 and then crossed to UAS-Notch , UAS- Gal4 (#50150); w ;GMR46B08-Gal4(#47361); pros ,ry (#11744; PresenilinRNAi or UAS-Nintra. Crosses were established and cultured at 18°C 82B π pros-lacZ); y,w; UAS-GFP.nls (#4775); w; FRT ,hs M (#2004; wild type); until adulthood. F1 female progeny of the appropriate genotype were aged UAS-dicer 2 (#24648); tub-Gal80TS(#7108); tub-Gal80TS (#7017); ry506 05151 19A 3-5 days at 18°C and then shifted to 29°C for 7 days. Global activation of the P{PZ} Dl /TM3 (#11651; Dl-lacZ); w, hsflp, tub-Gal80, FRT (#5133); intra k606 RNAi RNAi Notch pathway using the hs-N transgene was accomplished by providing y,w; esg (#10359); UAS-N (#7078); UAS-psn (#27681); UAS- two 30-min heat shocks at 37°C, one in the morning and the second at night. Nintra (#52008).w1118;npf-Gal4(Wu et al., 2003); Fer1-GFP (Flytrap nuc #G00188); w; actin5C FRT stop FRT lacZ ;UAS-flp(actin5C>lacZ;Struhl Histology 82B and Basler, 1993); y,w, UAS-GFP, hsflp; tub-Gal4, FRT , tub-Gal80/TM6B; Generation of whole-mount samples for immunostaining was performed as Su(H)GBE-Gal4 (Zeng et al., 2010); Su(H)GBE-lacZ (Furriols and Bray, previously described (Micchelli, 2014). Briefly, adult females were in fixed 55e11 19A 2001); esg-Gal4, UAS-GFP (Micchelli and Perrimon, 2006); N FRT / 0.5× PBS and 4% EM-grade formaldehyde (Polysciences) overnight, 19A FM7 (Kidd et al., 1983); w, hsflp, tub-Gal80, FRT ; UAS-GFP, UAS-lacZ; washed in 1× PBS, 0.1% Triton X-100 (PBST) for a minimum of 2 h, and TS tub-Gal4; w; esg-Gal4, UAS-GFP, tub-Gal80 (Micchelli and Perrimon, incubated in primary antisera (see below) overnight. Samples were washed TS rev10 82B rx106 82B 2006; esg ); Dl FRT (Haenelin et al., 1990); Ser FRT (Thomas and incubated in secondary antibodies for 3 h, washed again in PBST and k606 82B et al., 1991); y,w, UAS-GFP, hsflp; esg ; tub-Gal4, FRT ,tub-Gal80/ mounted in Vectashield+DAPI. All steps were completed at 4°C. TM6B; hs-Nintra (Struhl et al., 1993). For additional information, see http://flybase.org. Antisera Chicken anti-GFP (Abcam, #ab13970; 1:10,000); mouse anti-β-Gal (DSHB, Characterization of neuropeptide distribution #40-1a; 1:100); mouse anti-Pros (DSHB, #MR1A; 1:100); mouse anti-Brp Characterization of adult midgut neuropeptide distribution was carried (DSHB, #nc82; 1:10); mouse anti-Cut (DSHB, #2B10; 1:100); mouse anti- out in wild-type flies (w1118 or Canton-S). We present the results of AstA (DSHB, #5F10; 1:20); rabbit anti-β-Gal (Cappel, #08559761; 1:4000); neuropeptide distribution in newly eclosed flies selected from low-density rabbit anti-Tk (1:500, a generous gift from Dr Dick Nässel, Stockholm cultures, aged for 5 days at 25°C and dissected directly in fixative. University, Sweden) rabbit anti-Npf (1:750, a generous gift from Dr Paul Taghert, Washington University, USA); rabbit anti-Phm (1:250, a generous gift from Dr Paul Taghert); rabbit anti-AstB (1:500, a generous gift from Drs Directed cell lineage tracing Paul Taghert and Jan Veenstra, Université de Bordeaux, France); rabbit anti- Directed tracing of adult endocrine cell lineages was performed by crossing AstC (1:500, a generous gift from Dr Jan Veenstra); rabbit anti-DH31 (1:500, specific Gal4 driver lines to the w; actin5C FRT stop FRT lacZ; UAS-flp a generous gift from Dr Jan Veenstra). Alexa Fluor-conjugated secondary lineage tracer and culturing at 25°C. Neuropeptide Gal4 lineage tracing antibodies (Molecular Probes, #A-11039, #A-11029, #A-11031, #A-21236, experiments were analyzed as newly eclosed virgins (day 0) and then 4 and #A-11034, #A-11036, #A-21245; 1:2000). 10 days after eclosion under baseline conditions; they were also analyzed 8 days after eclosion following a 24 h Pe (OD ) infection. Conditional lineage 5 Microscopy and imaging tracing was performed by combining either neuropeptide Gal4 or Su(H)-Gal4 Samples were analyzed on a Leica DM5000 or Leica TCS SP5 confocal lines with tub-Gal80TS. The resultant flies were then crossed to the w; actin5C microscope. Images were processed for brightness and contrast in FRT stop FRT lacZnuc;UAS-flplineage tracer. Crosses were established and Photoshop CS (Adobe). cultured at 18°C until adulthood. F1 female progeny were aged 3-5 days at 18° C and were then shifted to 29°C for defined periods. Su(H)-Gal4TS lineage- Acknowledgements tracing experiments were analyzed 10-12 days following the shift to 29°C and We wish to acknowledge the collegiality of members of the fly community who freely TS compared with unshifted controls grown at 18°C for 10 days. Su(H)-Gal4 shared reagents. We are indebted to Drs D. Nässel, P. Taghert and J. Veenstra for lineage-tracing experiments were also analyzed following challenge by heat generous gifts of very limited antibodies, without which this study would simply not stress or ad libitum exposure to Pe. For heat challenge, adults were grown at have been possible. We also acknowledge Joseph Burclaff for initial screening 29°C for 5 days, heat-shocked (37°C water bath, 45 min in the morning, plus and characterization of neuropeptide Gal4 lines. Finally, we thank members of the 45 min at night) and cultured for an additional 5 days (10 day ‘chase’). An Micchelli lab for constructive discussions and comments on the manuscript. additional heat challenge protocol was also tested. Adults were grown at 29°C for 7 days, heat-shocked (37°C water bath, 45 min in the morning, plus 45 min Competing interests at night) and cultured for an additional 7 days (14 day ‘chase’). For bacterial The authors declare no competing financial interests. challenge, adults were grown at 29°C 7 days, exposed to Pe (24 h on Pe OD20- laced food vials at 29°C) and cultured for an additional 7 days (15 day Author contributions R.B.-E. performed the experiments. R.B.-E. and C.A.M. wrote the manuscript. ‘chase’). Funding Mosaic analysis This work was supported by grants from the American Cancer Society and the Positively marked cell lineages were generated in female midguts using National Institutes of Health [NIH RO1 DK095871 to C.A.M.]. Deposited in PMC for the MARCM system. Fly crosses were cultured on standard media release after 12 months. DEVELOPMENT

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