RESEARCH ARTICLE STEM CELLS AND REGENERATION

Generation of enteroendocrine cell diversity in midgut stem celllineagesRyan Beehler-Evans and Craig A. Micchelli*

ABSTRACTThe endocrine system mediates long-range peptide hormonesignaling to broadcast changes in metabolic status to distant targettissues via the circulatory system. In many animals, the diffuseendocrine system of the gut is the largest endocrine tissue, with thefull spectrum of endocrine cell subtypes not yet fully characterized.Here, we combine molecular mapping, lineage tracing and geneticanalysis in the adult fruit fly to gain new insight into the cellular andmolecular mechanisms governing enteroendocrine cell diversity.Neuropeptide hormone distribution was used as a basis to generate ahigh-resolution cellular map of the diffuse endocrine system. Ourstudies show that cell diversity is seen at two distinct levels: regionaland local. We find that class I and class II enteroendocrine cells canbe distinguished locally by combinatorial expression of secretedneuropeptide hormones. Cell lineage tracing studies demonstratethat class I and class II cells arise from a common stem cell lineageand that peptide profiles are a stable feature of enteroendocrine cellidentity during homeostasis and following challenge with the entericpathogen Pseudomonas entomophila. Genetic analysis shows thatNotch signaling controls the establishment of class II cells in thelineage, but is insufficient to reprogram extant class I cells into class IIenteroendocrine cells. Thus, one mechanism by which secretory celldiversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages.

KEY WORDS: Drosophila, Diffuse endocrine system, Neuropeptide,Stem cell, Notch

INTRODUCTIONOn 16 January 1902, Ernest Starling observed a striking increase inpancreatic secretion when he prepared lysate from a segment offreshly excised intestinal mucosa rubbed with sand and weak HCl,filtered it through cotton wool and injected the extract into thejugular vein of an anesthetized dog (Bayliss and Starling, 1902).Documentation of this novel ‘reflex’ marked the inception ofendocrinology as a field and implicated the intestinal epithelium asa potent source of peptides capable of acting over a great distance viathe circulatory system to profoundly alter physiology. We now knowthat the gut epithelium is a rich source of secreted hormones thatoriginate from specialized enteroendocrine cells and comprise adiffuse endocrine system distinct from the well-studied glandularendocrine system (Engelstoft et al., 2013; Schonhoff et al., 2004). Gutendocrine cells are embedded in the barrier epithelium and mediateluminal chemosensitivity (Furness et al., 2013; Reimann et al., 2012).In response to environmental stimuli, peptides secreted from the gut

epithelium act broadly on endocrine, nervous and immune systems toencode adaptive physiologic responses. Such peptides (also calledneuropeptides) belong to distinct families, such as allatostatins,diuretic hormones and tachykinins (Bendena et al., 1999; Gäde, 2004;Nässel andWegener, 2011; Van Loy et al., 2010). Knowledge differswidely with respect to each peptide family member and theirexperimentally defined roles within a given species. However,neuropeptide hormones have been implicated in processes such asgrowth control, muscle activity, feeding behavior, osmotic balance,reproduction and epithelial secretion. The prodigious number ofenteroendocrine cells and their varied neuropeptide expressioncombine to make the gut the largest endocrine organ in the body.And yet, the mechanisms underlying endocrine diversity in thiscentral endocrine tissue have not been fully understood.

The invertebrate gastrointestinal (GI) tract, like its mammaliancounterpart, contains a diffuse endocrine system providing atractable experimental model to investigate the basis of enteroe-ndocrine diversity. This population of cells can be easily detectedalong the entire anterior-posterior (A/P) axis of the adult midgutwith the pan-enteroendocrine marker Prospero (Pros). Adultenteroendocrine cells are continuously maintained by stem celldivisions that occur in physiologically and functionally distinctregions of the midgut (Micchelli and Perrimon, 2006; Ohlstein andSpradling, 2006; Strand and Micchelli, 2011). Genomic analysis atthe organ level has shown that a wide variety of neuropeptides andtheir cognate receptors, many of which are conserved, are expressedboth within the gut and in the periphery (Marianes and Spradling,2013; Buchon et al., 2013; Veenstra et al., 2008; Veenstra, 2009;Veenstra and Ida, 2014). It is also now possible to specificallyisolate enteroendocrine cells and to obtain expression profiles at thecellular level (Dutta et al., 2013). Moreover, experimental means tochallenge the barrier epithelium with pathogenic or nutritionalinsults are now available and combine readily with existingmolecular genetic tools (Vodovar et al., 2005; Lee and Micchelli,2013; Piper et al., 2014). Thus, the GI tract of Drosophila providesan unsurpassed experimental model to investigate aspects of diffuseendocrine system biology.

Studies of adult Drosophila enteroendocrine cells have focusedon the molecular events that determine whether a newly formedintestinal progenitor, called enteroblast, will adopt the secretory(endocrine) or absorptive (enterocyte) cell fate (Micchelli andPerrimon, 2006; Ohlstein and Spradling, 2006). The idea that Notchsignaling might control this decision was first suggested followingthe observation that high levels of a transcriptional reporter forNotch could be detected in the enteroblast (Micchelli and Perrimon,2006). Subsequent functional studies showed that the Notch ligandDelta can be detected at high levels in the neighboring intestinalstem cell and that Delta is both necessary and sufficientto distinguish the endocrine and enterocyte fate (Beebe et al.,2010; Ohlstein and Spradling, 2007). Together, these observationsled to a simple model in which stem cells signal to enteroblasts toReceived 1 July 2014; Accepted 15 December 2014

Washington University School of Medicine, Department of Developmental Biology,660 South Euclid Avenue, St Louis, MO 63110, USA.

*Author for correspondence (micchelli@wustl.edu)

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influence cell fate: low levels of Notch transduction in theenteroblast promote the enteroendocrine differentiation and highlevels of Notch transduction in the enteroblast promote theenterocyte differentiation. This asymmetry between stem cell anddaughter cell is reinforced by segregation of Sara endosomes intothe newly formed enteroblast (Montagne and Gonzalez-Gaitan,2014). However, precisely when the specifying Notch signalingevent occurs remains an open question (Perdigoto et al., 2011).Transcription factors of the Enhancer of split and achaete-scutecomplexes have both been shown to control endocrinedifferentiation (Bardin et al., 2010). More recently, Slit/Robosignaling has been implicated in enteroendocrine specification(Biteau and Jasper, 2014). Possible interactions between Notch andRobo 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 adultDrosophila and to gain insight into the molecular mechanismsunderlying this process. Here, we focus on the posterior midgut anduse differential neuropeptide expression as a criterion to mapenteroendocrine cell types in situ with great spatial resolution. Weshow that even immediately adjacent endocrine cells alongthe midgut express different combinations of neuropeptides,dividing the diffuse endocrine system into class I and class IIendocrine cells. Directed cell lineage-tracing studies show that, oncean enteroendocrine cell subtype is established, it is stablymaintained in the adult, even following environmental challenge.Thus, diversity of endocrine cells is seen at two levels: regional andlocal. We demonstrate that individual gut stem cells generate pairsof class I and class II enteroendocrine cells displaying distinctneuropeptide profiles. Moreover, the precise combination ofneuropeptide expression depends on stem cell position within themidgut. Finally, our analysis shows that Notch signaling is initiallyrequired to generate class II enteroendocrine cell diversity inposterior midgut stem cell lineages, but is dispensable for bothunderlying regional identity and neuropeptide expression in extantendocrine cells. We conclude that local endocrine diversity isgenerated in the diffuse endocrine system during adult homeostasisthrough cell-cell signaling interactions within individual stem celllineages.

RESULTSNeuropeptide expression distinguishes adultenteroendocrine cells in the posterior midgutThe adult Drosophila midgut epithelium is a rich source ofsecretory neuropeptide hormones, showing elevated levelsof Allatostatin A (AstA), Allatostatin B (AstB), AllatostatinC (AstC), Neuropeptide F (Npf ), Diuretic hormone 31 (DH31)and Tachykinin (Tk) (Veenstra et al., 2008; Reiher et al., 2011;Veenstra and Ida, 2014). Neuropeptide expression subdivides thegut into broad regions along the A/P axis, which are evident withregards to both anatomical and molecular markers (supplementarymaterial Fig. S1A-N; Table S1) (Veenstra et al., 2008; Veenstra andIda, 2014). For example, the posterior midgut is divided into twoprimary neuropeptide-expressing regions that we refer to as region1 and region 2, respectively (Fig. 1A; supplementary materialFig. S1A,B, Fig. S2A-F; Table S1). A panel of transgenicmolecular markers have been used to subdivide the posteriormidgut into regions (Marianes and Spradling, 2013; Buchon et al.,2013). However, careful comparison of relevant marker strainswith neuropeptide antisera revealed segmental boundaries thatwere distinct (supplementary material Fig. S2A-F). Therefore,for the purpose of this study, which focuses primarily on the

peptide-rich posterior midgut, we use the ‘region 1’ and ‘region 2’designations to refer directly to native domains of peptideexpression in the adult midgut.

Fig. 1. Diversity of adult enteroendocrine cells is both regional and local.(A) Regional differences in peptide hormone expression distinguish segmentsof the adult posterior midgut (shading). (B) Within each region, localdifferences in peptide hormones are detected among endocrine cell pairs.Class I and II enteroendocrine cells are distinguished by peptide antisera andGal4 lines. Yellow indicates Notch-dependent cell fates. DH, DH31.(C-H) Peptides are differentially detected in Pros+ cell pairs. (C) Anti-AstA, red;anti-Pros, red. (D) Anti-AstB, red; anti-Pros, green. (E) Anti-AstC, red; anti-Pros, green. (F) Anti-DH31, red; anti-Pros, green. (G) Anti-Npf, red; anti-Pros,green. (H) Anti-Tk, red; anti-Pros, green. (I) Pairs of endocrine cells expressdifferent peptides. Anti-AstA, red; anti-Tk, green. (J) At the transition betweenregion 1 and 2, AstA increases posteriorly, whereas AstB decreasesposteriorly. Anti-AstA and anti-AstB, white; Tk>GFP, green. Scale bars: 10 µm.

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Adult stem cells maintain the differentiated epithelial lining ofthe midgut (Micchelli and Perrimon, 2006; Ohlstein andSpradling, 2006). This monolayer consists primarily of largeabsorptive enterocyes, but also includes ∼1000 (∼10%) secretoryenteroendocrine cells that can be identified by the pan-endocrinemarker Pros. However, it has been unclear whether neuropeptideexpression in the midgut is restricted only to endocrine cells, orthe extent to which individual cells might express specificcombinations of particular neuropeptides. To determine whethersecretory neuropeptides are expressed in Pros+ cells, weconducted a series of double-labeling experiments on five-day-old adult midguts. Our studies show that distinct subsets of Pros+

cells also stain positive for AstA, AstB, AstC, DH31, Npf andTk when assayed in a pairwise manner (Fig. 1C-H). Initialcharacterization indicated that, with the exception of AstC inregion 1, only one of the two adjacent Pros+ cells stained positivefor the peptide assayed. This impression was confirmed by scoringthe percentage of Pros+ cells within a given region that alsoexpressed a particular neuropeptide (Table 1). Values ranged from48 to 52%, or approximately half of the Pros+ cells. Neuropeptideexpression was specific to endocrine cells, with no obviousexpression detected above background levels elsewhere in the gutepithelium. These results were independently confirmed usingspecific neuropeptide Gal4 driver lines, which we found tobe 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 endocrinecells, and in all but one case, labels half of the Pros+ cells in aparticular 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). Acomprehensive series of double- and triple-staining studies,employing both antibodies and Gal4 lines, was performed. Thisanalysis showed that in region 1, for example, class I enteroendocrinecells are AstB+, AstC+, whereas class II enteroendocrine cells areAstC+ (supplementary material Fig. S3E,F). In region 2, classI enteroendocrine cells are AstA+, AstC+ (supplementary materialFig. S3C,D,G,H), whereas class II enteroendocrine cells are DH31+,Tk+ (supplementary material Fig. S3I,J,M,N). To determinewhich fraction of Pros+ cells express either class I or class IIneuropeptide markers we simultaneously labeled AstC+, Tk+ andPros+ cells (Table 1). Using combinations of either antisera orGal4 lines revealed that the vast majority (>95%) of Pros+ cellsalso express neuropeptides. A transition zone between region 1and region 2 was identified, where neuropeptide protein isdetected in a graded manner within class I endocrine cells (Fig. 1J;supplementary material Table S1). We note that pairs of class Iand class II enteroendocrine cells were also detected in the

anterior and middle regions of the adult midgut (Fig. 1G;supplementary material Fig. S1; Table S1). Thus, precisecombinations of secretory neuropeptides can be used todistinguish each cell within an enteroendocrine pair 5 days aftereclosion.

Neuropeptide expression is a fixed characteristic ofenteroendocrine cell subtypesNeuropeptides activelymodulate organismal physiology. It is thereforepossible that established cellular profiles of peptide expressionassociated with a class of enteroendocrine cells can change duringadulthood. Alternatively, peptide expression might be a fixedcharacteristic of endocrine cell subtypes. To test these possibilities,we conducted directed cell lineage-tracing experiments using specificneuropeptideGal4 driver lines (Fig. 2). Class I enteroendocrine cells inregion 2 of the posterior midgut were genetically labeled by crossing

Table 1. Distribution of neuropeptides in Prospero+ cells

Antigen Neuropeptide+ Pros+Neuropeptide+/Pros+ ratio (%) Region n (guts)

AstA 106 206 51 2 6AstB 42 88 48 1 3AstC 261 269 97 1 6DH31 196 383 51 2 6Npf 245 500 49 Anterior+CC 6Tk 252 525 48 2 6AstC/Tk 749 792 95 1+2 6GFP* 1487 1557 96 1+2 8

*AstC-Gal4/Tk-Gal4×UAS-GFP; CC, Copper cell region.

Fig. 2. Peptide expression stably distinguishes class I and IIenteroendocrine cells. (A-H) Directed lineage tracing of class I and IIenteroendocrine cells using actin5C>lacZ; UAS-flp. Anti-β-Gal, red; anti-AstA/anti-DH31, green. (A,C,E,G) AstA-Gal4. (B,D,F,H) Tk-Gal4. (A,B) Gal4 linesare not detected in newly eclosed adult midguts. (C,D) 4 days followingeclosionGal4 driversmark endocrine subtypes. Marked cells are distinguishedfrom their neighbors with neuropeptide antisera. (E,F) 10 days followingeclosion. (G,H) Challenge with Pe. Scale bar: 10 µm.

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the AstA-Gal4 to the actin5C FRT stop FRT lacZnuc; UAS-flp lineagetracer (AstA>Trace; Fig. 2A,C,E,G). The AstA-Gal4 driver line wasfirst detected in theadultmidgut 4 days following eclosion (Fig. 2A,C).Ten days following eclosion, midguts were stained to identify DH31+

cells, a marker of class II enteroendocrine cells in region 2 of theposterior midgut. We observed that cells labeled by AstA-Gal4 wereDH31−, although DH31 staining was readily detected in adjacentendocrine cells (Fig. 2E). In a complementary experiment, we labeledclass II enteroendocrine cells in region 2 using Tk-Gal4 (Fig. 2B,D,F,H). The Tk-Gal4 driver line was also first detected in the adult midgut4 days following eclosion (Fig. 2B,D). Ten days following eclosion,we stained forAstA+, a neuropeptide thatmarks class I enteroendocrinecells in region 2. We observed that cells labeled by Tk-Gal4 wereAstA−, although AstA+ cells were readily detected in adjacentendocrine cells (Fig. 2F). To further document these observations wecounted the number of lacZ+, DH31+ and lacZ+, AstA+ cells present inadult midguts at 0, 4 and 10 days after eclosion, and again 24 hfollowing challenge with the Gram-negative enteric pathogenPseudomonas entomophila (Pe) (Fig. 2G,H; Table 2). This analysisshows that AstA-Gal4 and Tk-Gal4 driver lines mark endocrine cellsubtypes that remain DH31− or AstA− during adult homeostasis, aswell as following pathogenic challenge. Taken together, theseexperiments suggest that the class I and class II neuropeptide profilesremain stable in enteroendocrine cells during adult gastrointestinalhomeostasis as well as following pathogenic challenge. Thus,neuropeptide expression provides a reliable means of distinguishingdistinct enteroendocrine cell subtypes in the adult midgut.

Class I and class II enteroendocrine cells are lineally relatedThe presence of enteroendocrine cells with distinct neuropeptideexpression profiles raised the question whether such cells mightarise from a common cell lineage or whether they are lineallydistinct (Fig. 3A). To distinguish these possibilities, weconducted cell lineage-tracing studies. Wild-type lineages werelabeled using the MARCM system (Lee and Luo, 1999) andstained to identify enteroendocrine subtypes. We focused first onregion 2 of the posterior midgut, where simultaneous detection ofclass I and II enteroendocrine cells was possible. Here, markedlineages were found to span both class I and II endocrine cellsubtypes (Fig. 3B-E). To further characterize the distribution ofendocrine cell subtypes, the number of AstA+ and Tk+ cells perclone was scored for each marked lineage recovered (Table 3).The total number of AstA+ and Tk+ cells per clone ranged from 0to 4. In every case in which a marked lineage contained two ormore endocrine cells, we scored the presence of both AstA+ andTk+ enteroendocrine cells. We conclude that in region 2 of theposterior midgut, class I and II enteroendocrine cell pairs arisefrom a common cell lineage.

Characterization of wild-type lineages was also performed inregion 1 of the posterior midgut, yielding similar results. However,analysis of the region 1 was limited by the lack of markers tosimultaneously distinguish class I and class II enteroendocrinecells (e.g. Fig. 1B and Fig. 3F,G; supplementary materialTable S1). Thus, we scored the fraction of Pros+ cells that werealso AstB+ (Fig. 3H,I; Table 4). We predicted that half of the Pros+

enteroendocrine cells present in each clone would be AstB+. Wefound that marked lineages recovered in region 1 containedbetween 0 and 3 Pros+ cells. However, among the clones thatcontained two Pros+ cells, 48% fit this criterion (Table 4). Insummary, nearly half of the wild-type clones observed in region 1were consistent with a model in which class I and II cells arisefrom a common lineage, as shown for region 2. It is likely that themethod of clone scoring used for analysis in region 1 enriched fora set of early clones containing less differentiated cells (i.e. Pros+

only). By contrast, the analysis in region 2 was biased toward lateclones identified by the presence of highly differentiatedendocrine cells (i.e. AstA+, Tk+). Taken together, analysis ofwild-type cell lineages supports a general model in which class Iand class II endocrine cell pairs of the posterior midgut arise froma common stem cell lineage.

Class II enteroendocrine cells arise from a Su(H)-Gal4expressing progenitorThe choice of cell fate between absorptive enterocytes and secretoryendocrine cells in the midgut is controlled by the Notch signalingpathway (Bardin et al., 2010; Beebe et al., 2010; Micchelli andPerrimon, 2006; Ohlstein and Spradling, 2006, 2007; Perdigotoet al., 2011). Activation of Notch signaling in stem cell daughters,called enteroblasts, promotes enterocyte formation, whereas reducedNotch signaling leads to the production of ectopic enteroendocrinecells. This model predicts that directed cell lineage-tracing ofprogenitors transducing high levels of Notch signaling should labelonly enterocytes. We tested this prediction using a pulse/chaseexperiment and the conditional Su(H)-Gal4, tub-Gal80TS strain tolabel a fraction of adult cell lineages with actin5C FRT stop FRTlacZnuc; UAS-flp under both baseline conditions and followingenvironmental challenge (Table 5; Fig. 4A-D). As predicted,enterocytes were readily labeled by this procedure when cellmarking was initiated, following a shift to the non-permissivetemperature (Fig. 4A-D). However, quite unexpectedly, we alsorecovered labeled Tk+ class II endocrine cells at a frequency of0.65% under baseline conditions (Table 5; Fig. 4A). Similarly, weobserved labeled DH31+ cells, a secondmarker correlated with classII endocrine fate, following environmental challenge (Table 5;Fig. 4C). By contrast, Tk+ class II cells were never labeled inunshifted control experiments; nor were AstA+ class I cells labeled at

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)

0 0 0 0 54 0 54 0 410 1 197 <1 78* 0 161 0 6

Tk>Trace Timepoint (days) AstA+β-Gal+ β-Gal+ AstA+β-Gal+/β-Gal+ ratio (%) n (guts)

0 0 0 0 64 0 146 0 710 0 244 0 88* 1 161 <1 7

* 24 h Pe challenge.

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the non-permissive experimental temperature (Table 5). Thissuggested that Notch signaling might be active specifically in theclass II enteroendocrine cell lineage. Consistently, Notch pathwayreporters were also detectable in Tk+ cells (Fig. 4E-H). From this, weconclude that DH31+ and Tk+ class II enteroendocrine cells arisefrom a Su(H)-Gal4-expressing progenitor. These findings prompteda series of experiments to directly test the function of Notch

signaling in the generation of regional diversity among midgutendocrine cell subtypes.

Class II enteroendocrine cells are established by Notch/Delta signalingNotch loss in the intestinal stem cell lineage leads to the productionof ectopic Pros+ cells, suggesting the formation of enteroendocrinetumors (Micchelli and Perrimon, 2006; Ohlstein and Spradling,2006). Yet, the full extent of endocrine cell differentiation in thesetumors is unclear. Therefore, we first confirmed and extendedprevious studies by generating nullNotch clones and monitoring theendocrine fate using an expanded panel of endocrine cell markers,including the pan-endocrine marker Pros; the ELKS family proteinBruchpilot (Brp), which supports calcium-dependent secretoryvesicle release; and cell morphology. In addition, we examined thelevels of Peptidylglycine α-hydroxylating monoxygenase (Phm).Phm is an enzyme that catalyzes the rate-limiting step in C-terminalα-amidation, a necessary step for the production of activeneurosecretory peptides from pro-forms of the peptides. We foundthat Notch loss led to ectopic cells displaying the characteristicmorphology of mature enteroendocrine cells with polarized cellularprocesses projecting toward the gut lumen (Fig. 5A). In addition,these cells were Pros+, Brp+ and Phm+ (Fig. 5B-D). Thus, weconclude that the ectopic cells that arise in Notch mutant lineagesexhibit multiple features of well-differentiated endocrine tumors.

By contrast, Notch loss was found to differentially affect thedistribution of specific secretory neuropeptides. We examineddistinguishing markers for both class I and class II enteroendocrinecells in region 2 of the posterior midgut (Fig. 5E-T). Notch clones inregion 2 led to loss of both DH31+ and Tk+, whereas AstA+ cellsremained present in the lineage (Fig. 5E-L). We quantified thisphenotype and found that, in every case in which two or moreendocrine cells were present, clones retained AstA+ cells but lostTk+ cells (Table 3). Similar results were observed using additionalclass I and class II markers in the anterior midgut, copper cell regionand region 1 of the posterior midgut (Fig. 5M-T; supplementarymaterial Fig. S4; Table S4). Importantly, these findings were

Table 4. Analysis of neuropeptides in region 1 cell lineages

WT 0 Pros+ 1 Pros+ 2 Pros+ 3 Pros+

0 AstB+ 92 15 27 21 AstB+

– 9 28 22 AstB+

– – 3 33 AstB+

– – – 0

Genotype AstB+Pros+ Pros+AstB+Pros+/Pros+ ratio (%) n (guts)

WT 51 161 32 18N55e11 149 249 60 7

Table 3. Analysis of neuropeptides in region 2 cell lineages

WT 0 AstA+ 1 AstA+ 2 AstA+ ≥3 AstA+

0 Tk+ 31 8 0 01 Tk+ 5 10 1 02 Tk+ 0 2 1 0≥3 Tk+ 0 0 0 0

N55e11 0 AstA+ 1 AstA+ 2 AstA+ ≥3 AstA+

0 Tk+ 89 0 0 291 Tk+ 0 0 0 02 Tk+ 0 0 0 0≥3 Tk+ 0 0 0 0

Fig. 3. Class I and II enteroendocrine cells arise from a common lineage.(A) Wild-type class I (pink) and class II (red) endocrine cells are present inpairs distributed among enterocytes (gray). Alternate lineage models aredistinguished by cell-labeling studies (green). (B-I) Wild-typeMARCM lineages5 days after induction, dashed line. (B-E) Lineages spanning class I and IIentoendocrine cells, region 2. Anti-AstA, red; anti-Tk, green. Distinct endocrinesubtypes arise from a common lineage. (F-I) Marked lineages, region 1. Anti-Pros, white. (G) Anti-AstC, red. (I) Anti-AstB, red. Scale bar: 10 µm.

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independently confirmed using conditional Notch knockdown(supplementary material Fig. S5A-J). We note that, whereas Notchloss affected local neuropeptide profiles among endocrine pairs, itwas not associated with changes in underlying regional identity inneuropeptide expression (supplementary material Fig. S5K). Thus,Notch signaling is necessary to specify the class II endocrine cellsubtype in the adult midgut, without affecting neuropeptideprocessing enzymes or the cellular machinery implicated insecretory vesicle release.The Notch pathway can be activated by Delta and Serrate ligands.

As in the case of wild type, marked lineages lacking Serrateproduced 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 clonesexhibited greater heterogeneity in the progenitor marker esg-lacZthanNotch, which correlated with heterogeneity of the neuropeptidephenotype (Fig. 6G,H; supplementary material Fig. S6). Thus,Delta, but not Serrate, is necessary for establishing class IIenteroendocrine cells during adult midgut homeostasis.

Notch function is dispensable for endocrine cellmaintenanceNotch might function only in the establishment of endocrinediversity. Alternatively, Notch might function in both theestablishment and maintenance of endocrine diversity. Todistinguish these possibilities, we pursued a conditional, cell type-specific genetic loss-of-function approach. Conditional Notchknockdown was achieved in extant class II enteroendocrine cellsusing theUAS-GFP, tub-Gal80TS; Tk-Gal4 strain. However, despitestrong Notch knockdown with this construct (e.g. supplementarymaterial Fig. S5), no change in either DH31+ or AstA+ cells wasdetected (supplementary material Fig. S7A-D). This suggests thatNotch signaling is dispensable for maintenance of neuropeptideexpression in enteroendocrine cells.

Notch activation is sufficient to establish class IIenteroendocrine cellsWe tested whether Notch signaling is also sufficient to establishlocal differences in endocrine cell subtype. In a first test, weexamined the consequences of global Notch activation onendocrine cell identity by monitoring DH31+ cells at 6 and 24 hfollowing induction of an hs-Notchintra transgene (supplementarymaterial Fig. S8). To distinguish newly formed cells frompreviously differentiated cells, we performed the experiment inan esg>GFP background. As previously reported, we readilyobserved that Notch pathway activation promoted differentiationof esg>GFP+ into large enterocytes 24 h following induction(supplementary material Fig. S8A,C). We also observed a lowfrequency of DH31+ doublets under these conditions, which arenot typically seen in wild-type cell lineages (supplementary

material Fig. S8C,D; Table 3). This observation is consistent withthe idea that Notch activation is sufficient to promote class IIendocrine cell fate. The low frequency phenotype is predictedbecause endocrine progenitors are an order of magnitude rarerthan epithelial progenitors and because, under baselineconditions, proliferation of all progenitors is infrequent. Wereasoned that, if progenitor pools are first increased, using geneticmeans, and then Notch is ectopically activated, the phenotypeshould be detected with greater frequency. Therefore, in a secondtest of Notch sufficiency, conditional Presenillin knockdown withesg-Gal4; UAS-GFP, tub-Gal80TS was followed by induction of

Table 5. Analysis of Su(H)>Trace

Peptide Peptide+β-Gal+ β-Gal+Peptide+β-Gal+/β-Gal+ ratio (%)

‘Chase’(days)

n(guts)

Tk 0 9 0 10* 6Tk 11 1690 0.65 10-12‡ 5AstA 0 1141 0 10-12‡ 5Pros 5 731 0.68 10§ 5DH31 8 1214 0.71 14§ 10DH31 19 2693 0.71 15¶ 10

*Unshifted, unchallenged; ‡shifted, unchallenged; § shifted, challenged (heatstress); ¶shifted, challenged (Pe).

Fig. 4. Class II endocrine cells arise from a Su(H)-Gal4 progenitor.(A-D) Conditional lineage tracing of Su(H)-Gal4TS cells using actin5C>lacZ;UAS-flp, 12 days after induction. (A,B) Cellular products of Su(H)-Gal4TS

labeling, red/white; anti-Tk, green. Arrows indicate Tk+ cells derived from Su(H)-Gal4TS cells. Asterisks show unlabeled Tk+ cells. (B) Single channel.(C,D) Cellular products of Su(H)-Gal4TS, 7 days following exposure to Pe, red/white; anti-DH31, green. Arrows indicate DH31+ cells derived from Su(H)-Gal4TS cells. Asterisks show unlabeled DH31+ cells. (D) Single channel.(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. Notelower levels of the reporter in the Tk+ cells. (E) esg>GFP, green; anti-Tk, red.(F) Su(H)GBE-lacZ, white. (G,H) Dl-lacZ labeling, red/white; anti-Tk, green.(H) Single channel. Scale bars: 10 µm.

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the hs-Notchintra transgene. Under these conditions we observed arapid, robust and significant increase in the number of Pros+,DH31+ cells (Fig. 7A-F). Finally, we tested whether Notchactivation is sufficient to reprogram fully differentiatedenteroendocrine cell fate from class I to class II. However,conditional activation of Notchintra in AstA+ cells was insufficientto induce DH31 peptide in these cells (Fig. 7G,H). Altogether,these results suggest that Notch signaling is both necessary andsufficient to specify the class II endocrine cell subtype during

adult midgut homeostasis. However, once a class I endocrine cellis established, ectopic Notch activation is insufficient toreprogram subtype identity.

DISCUSSIONThe barrier epithelium of the gut is a unique biological interface. Itis here that a complex stream of continuously changing luminalstimuli are presented to the organism and must be converted intoappropriate physiologic and neuromodulatory states. The cells of

Fig. 5. Notch is necessary to establish class II endocrine cells. (A-T) Mosaic analysis ofNotch usingMARCM; representative clones are highlighted by dottedlines. (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, internalcontrols. (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.

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the diffuse endocrine system represent an early, if not the primary,level at which these changes are sensed and broadcast.We have usedDrosophila as a model system to address the molecular mechanismsby which cellular diversity in the diffuse endocrine system ishomeostatically maintained.

Organization of the adult enteroendocrine systemEndocrine cells can be characterized by the neuropeptides theysecrete. From this perspective, the adult diffuse endocrine systemexhibits both regional and local organization. We show thatneuropeptide expression in the adult midgut is restricted to cellspositive for Pros, a pan-endocrine marker defining the extent of thediffuse endocrine system. Among Pros+ cells, distinct segmentalregions of neuropeptide expression along the A/P axis are evident.Within each region, local differences in combinatorial neuropeptidedistribution distinguish class I and II endocrine cells. Our analysis

indicates that >95% of Pros+ cells express one or more distinctneuropeptides. We predict that the remaining ∼5% of Pros+ cellscorrespond either to newly formed endocrine cells in the process ofdifferentiating, dividing progenitors (see Biteau and Jasper, 2014;Zielke et al., 2014) or differentiated endocrine cells expressingpeptides other than those tested.

The mapping data presented here confirm and extend a rapidlygrowing body of work, which has provided essential insight into thedistribution of regulatory neuropeptides in Drosophila (Buchonet 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 significantdifferences in approach, methodology and scope.

Our systematic, combinatorial mapping strategy provides highcellular and temporal resolution of neuropeptide distribution in vivounder baseline conditions. A pairwise comparison of availableantiserawithGal4 driver lines suggests that patterns of neuropeptideexpression and protein distribution are similar, if not identical, in theadult midgut. Importantly, these validated reagents providepowerful and specific new tools for manipulating the endocrinesystem, as we demonstrate. Finally, directed cell lineage analysisshows that neuropeptide profiles are stable features of endocrinecells during both normal homeostasis and following challenge withthe enteric Gram-negative pathogen Pe.

In mammals, the classical view of the diffuse endocrine systemis the ‘one cell-one hormone’ dogma (Engelstoft et al., 2013;Schonhoff et al., 2004). However, recent studies in mouse andhuman have challenged this perspective (Egerod et al., 2012; Habibet al., 2012; Sei et al., 2011). It is now clear that the mammalian GItract exhibits not only regional diversity but also local diversity inthe combinatorial expression of neuropeptides in single endocrinecells of the diffuse endocrine system. For example, in the murineintestine, endocrine cells in the crypt express multiple endocrinehormones, but endocrine cells on the villus do not (Sei et al., 2011).Interestingly, in mammals, peptides that are coexpressed also appearto have overlapping or coordinated functions. Thus, broadcoexpression of secretory neuropeptides is a conserved feature ofthe diffuse endocrine system among metazoans.

Class I and class II endocrine subtypes arise from acommonstem cell lineageThe diffuse endocrine system differs from the glandular endocrinesystem in that it must be continuously renewed. The combinatorialdifferences among endocrine neuropeptides described here providean experimental basis to address how diversity is maintained in thediffuse endocrine system. We distinguish two possible models anddirectly demonstrate that endodermal stem cell lineages give rise toboth class I and class II endocrine cells from a single lineage. Thus,local diversity within the diffuse endocrine system is controlled byindividual stem cell lineages situated at distinct positions along thelength of the GI tract. Developmental studies have characterizedthe stepwise process of endocrine cell development (Micchelli et al.,2011; Takashima et al., 2011). Cell divisions occurring during latepupal stages initially establish pairs of gut enteroendocrine cells.Moreover, recent studies have implicated the gut neurosecretorypeptide Bursicon as a key peptide in the complex behavioral processof pupal eclosion (Scopelliti et al., 2014). The fact that cellulardiversity in the diffuse endocrine system is actively maintained byadult stem cell lineages suggests that endocrine cells have broadfunctional significance in maintaining many aspects of metabolichomeostasis throughout the course of adulthood. These crucialfunctions remain to be fully characterized.

Fig. 6. Delta is necessary to establish class II endocrine cells.(A-H) Analysis of Delta and Serrate lineages using MARCM, dotted line.(A,B) Serrate clone, 5 days after induction. (B) Anti-AstA, red; anti-Tk, green.(C,D) Delta clone, 5 days after induction. (D) Anti-AstA, red; anti-Tk, green.Asterisk shows Tk+ cell outside clone. (E,F) Delta clone, 10 days afterinduction. (F) Anti-AstA, red. (G,H) Delta clone, 10 days after induction.(H) Anti-AstA, red; esg-lacZ, white. Scale bars: 10 µm.

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Controlling cell type in the endocrine lineageSimultaneous detection of class I and II endocrine cells in markedendodermal lineages suggested that endocrine cell fate is controlledby signaling events within individual stem cell lineages. Su(H)-Gal4

progenitors were found to give rise to class II, but not class I,endocrine cells, under a wide variety of experimental conditions thatincluded baseline homeostasis and different environmentalchallenges, suggesting a key role for the Notch signaling pathwayin generating enteroendocrine diversity. We note that this findingdiffers significantly from another report, which concluded thatendocrine cells do not arise from a Su(H)Gal4+ progenitor (Biteauand Jasper, 2014). For example, in one experiment, we detected fivePros+ cells among a total of 731 labeled cells (10 days followinginduction, five posterior midguts), whereas Biteau and Jasper reportno Pros+ cells among 418 (4 days following induction, eight guts).This disparity is therefore most likely explicable by the short chaseperiod used and the relative rarity of endocrine progenitors in the gut.

Our subsequent functional characterization clearly showed thatNotch signaling is both necessary and sufficient to control celldiversity in the diffuse endocrine system. Although Notch loss leadsto well-differentiated endocrine tumors, they exhibit specific changesin the type of endocrine cells produced. By contrast, Notch functionwas dispensable in controlling underlying regional identity along thelength of the gut. Gaining insight into the process underlying regionalspecification represents an interesting line of future investigation.Although our analysis focused on the posterior midgut, where peptidediversity is greatest, our studies also indicate that similar requirementsforNotch in class II endocrine cells exist along the entire length of theGI tract. Finally, targeted manipulation of Notch signaling indifferentiated endocrine cells failed to alter endocrine identity,consistent with a model in which Notch signaling functionsspecifically in endocrine progenitors. Thus, Notch signaling playsat least two separable roles in controlling cell fate within the gut stemcell lineages: a previously known requirement in specifyingabsorptive enterocytes, and the newly described role in controllingthe diversity of secretory enteroendocrine cells along the length of theadult midgut demonstrated here (Fig. 7I, yellow).

Non-autonomous consequences of endocrine tumors are implicitby the very nature of the peptides these cells secrete. Indeed,carcinoid tumors are neuroendocrine malignancies that secrethormones, which cause debilitating symptoms in patients. Inaddition, loss of endocrine cell subtypes has been associated withmetabolic disruptions, including lipid absorption and glucosehomeostasis (Mellitzer et al., 2010). Our study suggests that these

Fig. 7. Notch activation is sufficient to establish class II enteroendocrinecells. (A-F) Analysis of Notch pathway activation on endocrine cell fate,8 days following conditional Presenilin knockdown using esgTS. Adults wereshifted to the non-permissive temperature for 7 days to induce Presenilinknockdown and then stained against DH31+ cells 24 h later, in the presenceor absence of global Notch pathway activation. (A,C,E) Anti-Pros, red.(A-D) Anti-DH31, green. (A,B) Presenilin knockdown leads to endocrinehyperplasia and a reduction in DH31+ cells. (C,D) Presenilin knockdownleads to endocrine hyperplasia and a rapid increase in DH31+ cells followingglobal Notch activation. Inset, DH31+ cells exhibit endocrine morphology.(E) Notch activation is sufficient to promote differentiation of esg+ cells tolarge enterocytes, green. (F) Quantitation. The average number of DH31+

cells per gut scored for each genotype (n=7-12 guts per genotype, error barsrepresent s.e.m.; ***P<0.0001). (G-H) Analysis of Notch pathway activation infully differentiated class I endocrine cells using AstA-Gal4TS. Adults wereshifted to the non-permissive temperature for 7 days. AstA>GFP, green;Anti-DH31, red. (G) Controls shifted to non-permissive temperature showconditional expression of GPF. (H) Notch overexpression in AstA+ cells doesnot cause expression of the class II marker DH31. (I) Schematic cell lineagediagram. Undifferentiated stem cell daughters (enteroblasts) give rise toeither 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.

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effects might result from either the loss of specific peptides oroverabundance of others in the diffuse endocrine system. Thus,further insight into how endocrine peptides are produced and howsecretion is controlled might have important consequences forunderstanding tumor pathophysiology and for developing targetedtreatments for metabolic disease.

MATERIALS AND METHODSFly stocksThe following fly stocks were used: from Bloomington Stock Center: w1118

(#39352; wild-type control); Canton-S (#1; wild-type control); w1118;GMR65D06-Gal4 (#39352; AstA-Gal4, this study); w1118; GMR61H08-Gal4 (#39283; AstC-Gal4, this study); w1118; GMR57F07-Gal4 (#46389;DH31-Gal4, this study); w1118; GMR61H07-Gal4 (#39282; called Tk-Gal4,this study); w1118; GMR50A12-Gal4/TM3 (#47618); w1118; GMR42C06-Gal4 (#50150); w1118; GMR46B08-Gal4 (#47361); pros10419, ry506(#11744;pros-lacZ); y,w; UAS-GFP.nls (#4775);w; FRT82B, hsπM (#2004; wild type);UAS-dicer 2 (#24648); tub-Gal80TS(#7108); tub-Gal80TS (#7017); ry506P{PZ} Dl05151/TM3 (#11651; Dl-lacZ);w, hsflp, tub-Gal80, FRT19A (#5133);y,w; esgk606 (#10359); UAS-NRNAi (#7078); UAS-psnRNAi (#27681); UAS-Nintra (#52008). w1118; npf-Gal4 (Wu et al., 2003); Fer1-GFP (Flytrap#G00188);w; actin5C FRT stop FRT lacZnuc; UAS-flp (actin5C>lacZ; StruhlandBasler, 1993); y,w,UAS-GFP, hsflp; tub-Gal4,FRT82B, tub-Gal80/TM6B;Su(H)GBE-Gal4 (Zeng et al., 2010); Su(H)GBE-lacZ (Furriols and Bray,2001); esg-Gal4, UAS-GFP (Micchelli and Perrimon, 2006); N55e11 FRT19A/FM7 (Kidd et al., 1983); w, hsflp, tub-Gal80, FRT19A; UAS-GFP, UAS-lacZ;tub-Gal4; w; esg-Gal4, UAS-GFP, tub-Gal80TS (Micchelli and Perrimon,2006; esgTS);Dlrev10 FRT82B (Haenelin et al., 1990); Serrx106 FRT82B (Thomaset al., 1991); y,w, UAS-GFP, hsflp; esgk606; tub-Gal4, FRT82B, tub-Gal80/TM6B; hs-Nintra (Struhl et al., 1993). For additional information, seehttp://flybase.org.

Characterization of neuropeptide distributionCharacterization of adult midgut neuropeptide distribution was carriedout in wild-type flies (w1118 or Canton-S). We present the results ofneuropeptide distribution in newly eclosed flies selected from low-densitycultures, aged for 5 days at 25°C and dissected directly in fixative.

Directed cell lineage tracingDirected tracing of adult endocrine cell lineages was performed by crossingspecific Gal4 driver lines to the w; actin5C FRT stop FRT lacZ; UAS-flplineage tracer and culturing at 25°C. Neuropeptide Gal4 lineage tracingexperiments were analyzed as newly eclosed virgins (day 0) and then 4 and10 days after eclosion under baseline conditions; they were also analyzed8 days after eclosion following a 24 h Pe (OD5) infection. Conditional lineagetracingwas performed by combining either neuropeptide Gal4 or Su(H)-Gal4lines with tub-Gal80TS. The resultant flies were then crossed to thew; actin5CFRT stop FRT lacZnuc; UAS-flp lineage tracer. Crosses were established andcultured at 18°C until adulthood. F1 female progenywere aged 3-5 days at 18°C and were then shifted to 29°C for defined periods. Su(H)-Gal4TS lineage-tracing experiments were analyzed 10-12 days following the shift to 29°C andcompared with unshifted controls grown at 18°C for 10 days. Su(H)-Gal4TS

lineage-tracing experiments were also analyzed following challenge by heatstress or ad libitum exposure to Pe. For heat challenge, adults were grown at29°C for 5 days, heat-shocked (37°C water bath, 45 min in the morning, plus45 min at night) and cultured for an additional 5 days (10 day ‘chase’). Anadditional heat challenge protocol was also tested. Adults were grown at 29°Cfor 7 days, heat-shocked (37°Cwater bath, 45 min in themorning, plus 45 minat night) and cultured for an additional 7 days (14 day ‘chase’). For bacterialchallenge, adultswere grown at 29°C 7 days, exposed toPe (24 h onPeOD20-laced food vials at 29°C) and cultured for an additional 7 days (15 day‘chase’).

Mosaic analysisPositively marked cell lineages were generated in female midguts usingthe MARCM system. Fly crosses were cultured on standard media

supplemented with yeast paste at 25°C. Newly eclosed females of theappropriate genotype were aged 3-5 days prior to clone induction. Cloneswere induced by placing vials in a 37°Cwater bath for 30-40 min. Flies wereheat-shocked 2-3 times within a 24 h period.

Pe infectionFlies were infected ad libitum with Pe. Infected flies were fed on foodsupplemented with 0.5 ml of Pe at OD5-OD20 in 5% sucrose. Mock-infectedflies were placed on food supplemented with 0.5 ml 5% sucrose. Flies wereinfected with Pe for 24 h and subsequently maintained at 29°C throughoutthe course of the experiment.

Conditional manipulation of Notch pathwayTo conditionally target progenitor cells or differentiated endocrine cellpopulations in the adult midgut, esg-Gal4, Tk-Gal4 or AstA-Gal4 was firstcombined with tub-Gal80TS and then crossed to UAS-NotchRNAi, UAS-PresenilinRNAi or UAS-Nintra. Crosses were established and cultured at 18°Cuntil adulthood. F1 female progeny of the appropriate genotype were aged3-5 days at 18°C and then shifted to 29°C for 7 days. Global activation of theNotch pathway using the hs-Nintra transgenewas accomplished by providingtwo 30-min heat shocks at 37°C, one in the morning and the second at night.

HistologyGeneration of whole-mount samples for immunostaining was performed aspreviously described (Micchelli, 2014). Briefly, adult females were in fixed0.5× PBS and 4% EM-grade formaldehyde (Polysciences) overnight,washed in 1× PBS, 0.1% Triton X-100 (PBST) for a minimum of 2 h, andincubated in primary antisera (see below) overnight. Samples were washedand incubated in secondary antibodies for 3 h, washed again in PBST andmounted in Vectashield+DAPI. All steps were completed at 4°C.

AntiseraChicken anti-GFP (Abcam, #ab13970; 1:10,000);mouse anti-β-Gal (DSHB,#40-1a; 1:100); mouse anti-Pros (DSHB, #MR1A; 1:100); mouse anti-Brp(DSHB, #nc82; 1:10); mouse anti-Cut (DSHB, #2B10; 1:100); mouse anti-AstA (DSHB, #5F10; 1:20); rabbit anti-β-Gal (Cappel, #08559761; 1:4000);rabbit anti-Tk (1:500, a generous gift from Dr Dick Nässel, StockholmUniversity, Sweden) rabbit anti-Npf (1:750, a generous gift from Dr PaulTaghert, Washington University, USA); rabbit anti-Phm (1:250, a generousgift fromDr Paul Taghert); rabbit anti-AstB (1:500, a generous gift fromDrsPaul Taghert and Jan Veenstra, Université de Bordeaux, France); rabbit anti-AstC (1:500, a generous gift fromDr JanVeenstra); rabbit anti-DH31 (1:500,a generous gift from Dr Jan Veenstra). Alexa Fluor-conjugated secondaryantibodies (Molecular Probes, #A-11039, #A-11029, #A-11031, #A-21236,#A-11034, #A-11036, #A-21245; 1:2000).

Microscopy and imagingSamples were analyzed on a Leica DM5000 or Leica TCS SP5 confocalmicroscope. Images were processed for brightness and contrast inPhotoshop CS (Adobe).

AcknowledgementsWewish to acknowledge the collegiality of members of the fly community who freelyshared reagents. We are indebted to Drs D. Nassel, P. Taghert and J. Veenstra forgenerous gifts of very limited antibodies, without which this study would simply nothave been possible. We also acknowledge Joseph Burclaff for initial screeningand characterization of neuropeptide Gal4 lines. Finally, we thank members of theMicchelli lab for constructive discussions and comments on the manuscript.

Competing interestsThe authors declare no competing financial interests.

Author contributionsR.B.-E. performed the experiments. R.B.-E. and C.A.M. wrote the manuscript.

FundingThis work was supported by grants from the American Cancer Society and theNational Institutes of Health [NIH RO1 DK095871 to C.A.M.]. Deposited in PMC forrelease after 12 months.

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Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.114959/-/DC1

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