483RESEARCH ARTICLE

INTRODUCTIONThe insect midgut, like the vertebrate intestine, is an endoderm-derived organ. Both the larval and adult Drosophila midguts arecomposed of a single layer of epithelial cells with two layers ofvisceral muscle (VM) wrapped outside. Inside the gut lumen, aperitrophic membrane separates the food from the intestinalepithelium. During both mammalian and insect embryonicdevelopment, Forkhead and GATA transcription factors playevolutionary conserved roles in the specification and subsequentmorphogenesis of the digestive tract (Stainier, 2005). Similarly,multiple signaling pathways, including the EGF, Wingless (Wnt),Dpp (TGFβ), Notch and Hedgehog pathways, are involved in theembryonic development of the Drosophila midgut and mammalianintestine (Sancho et al., 2004). In both systems, cross-talk betweenmesodermal cells and endoderm-derived epithelial cells in thegut primordium plays important roles during embryonic gutdevelopment (Stainier, 2005; Szuts et al., 1998).

Starting from embryonic development stage 11, the Drosophilamidgut epithelium consists of two distinct cell populations:differentiating midgut epithelial cells (larval enterocytes, ECs) andundifferentiated adult midgut progenitors (AMPs, also referred to asmidgut histoblast islets or midgut imaginal islets) (Hartenstein et al.,1992). In Drosophila embryos, AMPs can be marked by expressionof asense or by one of several lacZ- or Gal4-expressing enhancer-trapinsertions (Brand et al., 1993; Hartenstein et al., 1992; Hartenstein andJan, 1992). AMPs first appear as spindle-shaped cells localized to theapical surface of the midgut epithelium, but later migrate to the basalsurface of the epithelium where they remain throughout larvaldevelopment (Hartenstein and Jan, 1992; Technau and Campos-Ortega, 1986). Notch signaling has been shown to be involved in thedevelopment of Drosophila AMPs. In Notch mutant embryos, thenumber of AMPs in the midgut rudiment is strongly increased at theexpense of differentiated larval ECs (Hartenstein et al., 1992). Duringlarval development, the ECs grow in both size and ploidy by

undergoing several endocycles, reaching 64C (DNA content) by thewandering L3 stage (Lamb, 1982). The AMPs remain diploidthroughout larval development and appear as scattered islets of cells(hence the term ‘midgut histoblast islets’) in late-stage larval midguts.During pupal development, the ECs histolyze and a new adult midgutepithelium forms from the AMPs (Bender et al., 1997; Jiang et al.,1997; Li and White, 2003). Similar midgut progenitor cells have alsobeen found in other insect species (Corley and Lavine, 2006).

Recently, the adult Drosophila midgut has been shown to undergodynamic self-renewal, a process similar to that found in themammalian intestine/colon. Fly and mammalian gut homeostasisare both powered by intestinal stem cells (ISCs), and Notchsignaling plays similar roles in regulating their differentiation intomature gut cells (Fre et al., 2005; Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007; van Eset al., 2005). Thus, the Drosophila midgut may serve as a model tostudy gut homeostasis and the development of cancers, such ascolorectal carcinoma, that are directly associated with this dynamicprocess in humans.

Here we describe the development of the AMPs in Drosophilalarvae and pupae. We discovered that Drosophila AMPs divideextensively throughout larval development, and that theirproliferation can be separated into two distinct phases. During earlylarval stages, the AMPs divide and disperse to form islets throughoutthe midgut, but during late larval development the dividing AMPsare contained within these islets. Furthermore, our study revealedthat Drosophila EGFR signaling is both necessary and sufficient toinduce the proliferation of AMPs during larval development.

MATERIALS AND METHODSFly stocksUAS transgenesThe following were used: UAS-RasV12, UAS-RasV12S35, UAS-RasV12G37,UAS-Rafgof, UAS-λTOP, UAS-SEM, UAS-RafDN, UAS-Mkp3, UAS-sSpi,UAS-sKrn, UAS-Krn, UAS-grkΔTC and UAS-Vn1.2. UAS-RNAi transgeneswere obtained from the Bloomington Stock Center (Bloomington, IN,USA), the National Institute of Genetics Fly Stock Center (NIG, Japan) orthe Vienna Drosophila RNAi Center (VDRC, Austria). According toinformation from NIG and VDRC, all the RNAi lines used are specific tothe genes targeted (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp;VDRC, http://stockcenter.vdrc.at/control/main).

EGFR signaling regulates the proliferation of Drosophilaadult midgut progenitorsHuaqi Jiang and Bruce A. Edgar*

In holometabolous insects, the adult appendages and internal organs form anew from larval progenitor cells duringmetamorphosis. As described here, the adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgutprogenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs first disperse, but later proliferatewithin distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium.We find that signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceralmuscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz andKeren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during latelarval stages.

KEY WORDS: EGFR, Adult midgut progenitor (AMP), Intestinal stem cell (ISC)

Development 136, 483-493 (2009) doi:10.1242/dev.026955

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 FairviewAve N., Seattle, WA 98109, USA.

*Author for correspondence (e-mail: bedgar@fhcrc.org)

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MutantsFRT42D Egfrf1, FRT42D Egfr[CO], FRT82B Ras1Δc40b, spiA14 FRT40A,FRT42D shot[65-2], FRT42D shot[V104], vnP1749 FRT80B, rhodel1 FRT80B,Krn27-3-4, vnP1749, vnγ7, stet871 and ru1 were used (see FlyBase for furtherinformation: http://flybase.org).

Gal4/lacZ reportersesgGal4NP7397, spiGal4NP0261, MyoIAGal4NP0001 (NIG, Japan), rholacZAA69,rholacZX81, howGal424B and esglacZK00606 were used (Bloomington StockCenter).

Lineage analysisMARCM lineage analysisNewly hatched first instar [24 hours after egg deposition (AED)] or mid-third instar (96 hours AED) larvae of the correct genotype were heat shockedfor 45 minutes at 37°C. The midguts were then dissected from wanderingL3 larvae (120 hours AED) and analyzed.

Flp/Gal4 lineage analysisNewly hatched first instar larvae (24 hours AED) of the correct genotypewere heat shocked for 20 minutes at 37°C to induce clones and thendissected at various developmental stages and analyzed.

Enhancer trapsP-element enhancer traps with midgut expression were obtained fromseveral sources, including FlyView (University of Münster, Germany;http://flyview.uni-muenster.de) and GETDB (Gal4 Enhancer-Trap InsertionDatabase, NIG, Japan). We identified a number of enhancer traps showingreporter expression specifically in the AMPs, including one insertion in spi(NP0261) and several insertions in esg (NP0726, 7397 and 7399).esgGal4NP7397-driven GFP expression was used to mark the AMPs. We alsoidentified an enhancer trap in brush border Myosin IA (MyoIAGal4,NP0001) that drives GFP expression specifically in midgut ECs (Morgan etal., 1995).

Ectopic gene expressionWe generated inducible AMP-, EC- and VM-specific expression systems(esgGal4ts, MyoIAGal4ts and howGal4ts) by combining esgGal4NP7397,MyoIAGal4NP0001 or howGal424B (Hartenstein and Jan, 1992) withubiquitously expressed temperature-sensitive alleles of the Gal4 inhibitor,Gal80 (tubGal80ts; Bloomington Stock Center) and UAS-GFP.

Quantification of AMP clustersWe counted AMPs or AMP clusters marked by esgGal4-driven GFPexpression throughout the entire midgut during larval and pupaldevelopment. UAS-GFP, UAS-sSpi, UAS-sKrn or UAS-Krn were inducedin the AMPs starting from first instar larvae (24 hours AED) using theesgGal4ts system and the midguts were dissected from wandering L3 larvaeand the number of AMP clusters counted.

Generation of mutant AMP clonesClones of AMPs homozygous for Egfrf1, Egfr[CO], Ras1Δc40b, spiA14, vnP1749,rhodel1, shot[V104] or shot[65-2] were generated using the MARCM system(Lee and Luo, 2001). First instar larvae (24 hours AED) of the correctgenotype were heat shocked for 45 minutes at 37°C to induce clones. Larvaewere then dissected at 120 hours AED. The number of GFP-positive clustersin each clone was quantified; in most cases, clones from at least ten midgutswere counted.

RNA in situ hybridization and immunofluorescenceRNA in situ hybridization was performed as described (O’Neill and Bier,1994). Rabbit anti-dpERK (Cell Signaling) was used to detect MAPkinase activity in the midgut. Anti-Delta and anti-Prospero were obtainedfrom the Developmental Studies Hybridoma Bank and used to mark ISCsand enteroendocrine cells in the midgut. Rabbit anti-β-galactosidase(Cappel) was used to identify the esg-positive cells in an esglacZbackground. Rabbit anti-phospho-histone H3 (PH3, Upstate) was used toidentify dividing cells.

Quantitative real-time PCR (qRT-PCR)We used qRT-PCR to quantify levels of vn mRNA from midgut cDNA. FormRpL30 (reference gene) primers see Buttitta et al. (Buttitta et al., 2007); forvn: vn 5� primer, 5�-TCACACATTTAGTGGTGGAAG-3�; vn 3� primer, 5�-TCACACATTTAGTGGTGGAAG-3�. The relative expression of vn wasanalyzed on the Bio-Rad iQ5 system.

SectioningWandering L3 midguts were dissected in PBS and fixed in half-strengthKarnovsky’s fixative. Following dehydration, the tissues were embedded inEpon and sectioned at 1 μm. The sections were stained with Toluidine Blue.

RESULTSDrosophila AMPs divide extensively during larvaldevelopmentTo study the development of AMPs during larval development,we first looked for AMP markers. From existing collections ofDrosophila enhancer traps we identified Gal4 or lacZ enhancertraps that are expressed specifically in the AMPs. Among theseare Gal4 enhancer traps inserted in the escargot (esg) locus, whichencodes a member of the Snail family of transcription factors. esghas previously been shown to be expressed in imaginal discs andabdominal histoblast nests and is required there for maintainingcells in the diploid state during larval development (Hayashi et al.,1993). When combined with UAS-GFP, esgGal4 enhancer trapNP7397 drove GFP expression specifically in the larval AMPs(Fig. 1). Similar esg enhancer traps have been used to mark theadult ISCs and their daughter enteroblasts (Micchelli andPerrimon, 2006).

GFP expression driven by esgGal4 was detected in the AMPsscattered throughout the midgut of newly hatched larvae (24 hoursAED) (Fig. 1A, arrows). AMPs appeared as small diploid cells, andwere easily distinguishable from the large polyploid midgutenterocytes (ECs). The number of GFP-positive AMPs increasedduring early larval development (24-72 hours AED) (Fig. 1A,B);however, they remained dispersed. Cell contacts between pairedAMPs were readily observed in the early larval midgut (Fig. 1B,inset) and are likely to represent two daughter AMPs from theprevious division migrating away from each other. By mid-thirdinstar (96 hours AED), AMPs formed discrete 2- to 3-cell clusters(Fig. 1C), suggesting that they proliferate within individual isletsinstead of migrating away from each other. The AMPs continued toproliferate within these clusters (Fig. 1D), undergoing severalrounds of rapid proliferation to enlarge each cluster to 8-30 cells bythe onset of metamorphosis [0 hours after pupae formation (APF),~130 hours AED] (Fig. 1E).

These results do not support the idea that Drosophila AMPs arequiescent during larval development (Bodenstein, 1994). Instead,we observed that the AMPs proliferate extensively during larvaldevelopment, resulting in large increases in both the number(early larval stages) and size (late larval stages) of the AMPclusters. To further document this process, we analyzed AMPlineages by positively marking individual AMPs with GFP usingthe MARCM system (Lee and Luo, 2001). When clones wereinduced in first or second instar (24-48 hours AED), they allcontained multiple AMP clusters by the wandering L3 stage (120hours AED), and all cells in any GFP-positive cluster were GFP-positive (Fig. 2A-A�; Fig. 4A). However, when clones wereinduced in mid-third instar (96 hours AED), clusters mosaic forGFP were observed by the wandering L3 stage (120 hours AED)(Fig. 2B-B�). These results confirmed that the AMPs switch toproliferating within islets to form clusters by mid-third instar. Toquantify the number of divisions during the early proliferative

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phase, we counted the number of the marked AMP clustersencompassed by each clone. When induced in the newly hatchedfirst instar larvae (24 hours AED), the clones contained, onaverage, 7.5 GFP-positive clusters at the wandering L3 stage (120hours AED) (see Table S1 in the supplementary material). Thissuggests that the AMPs divide about four times during the earlylarval stages (note that only half of all the clusters generated byeach AMP were marked in the MARCM system). Since no mosaicAMP clusters were found in the late larval midgut when cloneswere induced at first or second instar, we propose that themajority, if not all, of the early larval AMPs disperse after eachcell division. We then counted the number of cells in each clusterat white prepupa formation (0 hours APF), when most of the AMPclusters have stopped proliferating. Each AMP cluster contained8 to greater than 30 cells. This indicates that the AMPs divide anadditional three to five times within a cluster, after the clusters areestablished. In total, the AMPs appear to divide seven to ten timesthroughout larval development.

Midgut development during early metamorphosisStaining for the division marker phospho-histone H3 (PH3)indicated that by white prepupa formation (0 hours APF), themajority of the AMPs had ceased their proliferation. Some AMPs inthe posterior midgut, however, did not cease proliferation until 4hours APF (data not shown). Meanwhile, the visceral muscles(VMs) contracted, and the larval midgut shortened itselfdramatically. At the same time, the AMP clusters fused to form anew midgut epithelium (Fig. 1F), while the larval midgut epitheliumbecame extremely compacted and was sloughed into the intestinallumen (Li and White, 2003). The midgut continued to contract andshorten, and by 12 hours APF it became a sac-like structurecontaining histolyzing larval epithelium inside the newly formedmidgut (Juhasz and Neufeld, 2008). Visceral muscles undergo aprocess termed ‘de-differentiation’, in which the muscle fibershistolyze; however, the muscle cells themselves do not die and willredifferentiate to form the adult midgut VM during latemetamorphosis (Klapper, 2000). During early metamorphosis (0-24

485RESEARCH ARTICLEDrosophila AMP development

Fig. 1. Development of Drosophila adult midgut progenitors (AMPs). AMPs were marked by GFP expression (green) driven by esgGal4NP7397.The numbers of GFP-positive AMPs, AMP clusters or adult intestinal stem cells (ISCs) are indicated in the appropriate panels. DNA is stained withDAPI (blue). (A) First instar larval midgut (24 hours AED). GFP was detected in the AMPs as individual diploid cells (arrows). (B) Early third instar larvalmidgut (72 hours AED). Larval enterocytes (ECs) undergo several rounds of endoreplication, enlarging the larval midgut. AMPs remain diploid andtheir numbers increase during the first two larval stages. However, they remain mostly dispersed as individual cells. Inset shows GFP expression thatis overexposed to show cell contacts between two neighboring AMPs. (C) Mid-third instar larval midgut (96 hours AED). AMPs form distinctive 2- to3-cell clusters. (D) Late third instar larval midgut (120 hours AED). AMPs continue to proliferate and enlarge the clusters. (E) White prepupa stage (0hours APF, ~130 hours AED). The size of the AMP clusters has increased further. (F) Prepupa stage (4 hours APF). The AMP clusters fuse to form anew midgut epithelium. Larval ECs (out of focal plane) are sloughed into the lumen and histolyze. (G,H) Early pupa stage (8 and 12 hours APF). Themajority of the cells in the new midgut epithelium gradually lose GFP expression, except for a few scattered cells that maintain strong GFPexpression. (I,J) Pupa stage (24 hours APF). The future adult ISCs are clearly identifiable by strong GFP expression (asterisks in I) and basallocalization in the epithelium (asterisks in J, cross-sectional view). GFP expression is lost in the rest of the cells in the new epithelium. Scale bars:20μm.

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hours APF), the majority of cells in the newly formed midgutepithelium gradually lost esgGal4-driven GFP expression, with theexception of a few scattered cells that maintained strong GFPexpression (Fig. 1F-I, asterisks). By 24 hours APF, these GFP-positive cells became basally localized in the new epithelium (Fig.1J, asterisks). As described below, we believe that these cells are thefuture adult midgut ISCs (see Discussion).

Around 24 hours APF, the esg-positive cells in the new adultmidgut epithelium started to proliferate (see Fig. S1A-A� in thesupplementary material). They continued to divide at 48 hours APFand increased in number (see Fig. S1B-B� in the supplementarymaterial). At 72 hours APF, the esg-positive cells continued todivide and some of them also expressed the enteroendocrine cellmarker Prospero (see Fig. S1C-C� in the supplementary material),indicating their capacity to differentiate. Whether these cells alreadybehaved as stem cells, which both self-renew and differentiate, wasnot determined. GFP-marked AMP Flp/Gal4 clones induced at earlylarval stages contained only 0-2 esg-positive cells at 24 hours APF(Fig. 2C-C�), but when these clones were scored later, in newlyeclosed adults, they contained both large apically localized ECs and

smaller, basally localized cells positive for the enteroendocrine cellmarker Prospero or the ISC marker Delta (Fig. 2D-D�,E-E�). Theseresults indicate that some of the AMPs become adult ISCs(Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006), andthat this transition occurs in the pupa. This was further supported byour observation that AMP clones induced in the larva persisted inthe adult midgut for at least 2 months (data not shown).

EGFR signaling stimulates AMP proliferationUsing the esgGal4ts system, we manipulated the activity of severalknown Drosophila signaling pathways specifically in the AMPs.Our tests included Wingless, Dpp, Hedgehog, Notch and EGFRsignaling components (see Table S2 in the supplementary material).Activation of EGFR/RAS/MAPK signaling in the AMPs was ableto drive their overproliferation during larval development.Compared with control midguts (Fig. 3A), in which the AMPsappeared as 2- to 3-cell clusters by mid-third instar (96 hours AED),the induction of activated Ras (Ras oncogene at 85D – FlyBase)(RasV12) in the AMPs led to the formation of much larger AMPclusters (Fig. 3B-D). Ectopic expression of RasV12 in the AMPs

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Fig. 2. Lineage analysis of the AMPs.(A-B�)Drosophila AMP clones induced using theMARCM system. Clones were induced at either firstinstar (24 hours AED, A-A�) or third instar (96 hoursAED, B-B�) and analyzed at the wandering L3 stage(120 hours AED). When induced at first instar, theclones appear as multiple marked clusters with all cellslabeled (A-A�), whereas clones induced late (at 96 hoursAED) were all confined to a single cluster that is mosaicfor GFP (B-B�). (C-E�) Pupal or adult AMP clones inducedusing the Flp/Gal4 system. Flp/Gal4 AMP clones wereinduced at first instar larval stage (24 hours AED) andanalyzed at 24 hours APF (C-C�) or from newly eclosedadults (D-E�). At 24 hours APF, each AMP clone contains0-2 esg-positive cells (C, arrows); the asterisk marks thehistolyzing larval midgut. In newly eclosed adults, themidgut contains enteroendocrine cells and ISCs; arrowsindicate cells within the clone that are positive forProspero (D) and Delta (E).

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throughout larval development resulted, by the wandering stage(120 hours AED), in a midgut comprising mostly esg-positiveAMPs (Fig. 3F) in which the intestinal lumen was occluded. Bycontrast, wild-type AMPs appeared as basally localized cell clustersin the midgut epithelium (Fig. 3E). The following evidencesuggested that EGFR signaling promoted AMP proliferationthrough activating the MAPK pathway. First, induction of RasV12S35,which preferentially activates the MAPK pathway, drove similarectopic proliferation of the AMPs as did RasV12 (see Table S2 in the

supplementary material), whereas expression of RasV12G37, whichpreferentially activates the Phosphotidylinositol 3 kinase (PI3K) orRal guanine nucleotide exchange factor 2 (RalGDS) pathway(Karim and Rubin, 1998; Prober and Edgar, 2002), had little effecton their proliferation (see Table S2 in the supplementary material).Second, ectopic expression of Dp110 (Pi3K92E – FlyBase; PI3K)had no detectable effect on AMP proliferation (see Table S2 in thesupplementary material). Third, increased proliferation of the AMPswas observed when activated Egfr (λTOP) (Queenan et al., 1997),gain-of-function Raf (Rafgof) (Brand and Perrimon, 1994) oractivated MAPK [sevenmaker (sem); rolled – FlyBase] (Martin-Blanco, 1998) was induced in these cells (see Table S2 in thesupplementary material). Fourth, expression of a dominant-negativeform of Raf (RafDN) (Roch et al., 1998) together with RasV12 gave aphenotype similar to that of RafDN alone (see Fig. S2E,F in thesupplementary material), and thus Raf is epistatic to Ras inregulating AMP proliferation. Fifth, expression of Mkp3, a negativeregulator of MAPK (Rintelen et al., 2003), did not affect AMPproliferation (see Fig. S2G in the supplementary material).Interestingly, however, Mkp3 did significantly suppress the AMPoverproliferation phenotype induced by RasV12 expression (see Fig.S2H in the supplementary material; compare with Fig. 3D).

EGFR signaling is required for AMP proliferationNext, we tested whether the EGFR/RAS pathway is required for thenormal proliferation of AMPs during larval development. Using thesame esgGal4ts system, we depleted crucial components of thepathway by expressing RNA inverted repeats (IR, RNAi) specific toDrosophila Egfr, Ras and Raf (pole hole – FlyBase) in the AMPs.As a control, UAS-driven RNAi directed against GFP was inducedin the AMPs using the esgGal4ts system. This treatment did notaffect AMP development (data not shown). When compared withcontrol midguts from white prepupa (0 hours APF), RNAi-mediateddepletion of each of these gene products in the AMPs throughoutlarval development significantly decreased both the number and sizeof the AMP clusters (see Fig. S2A-D and Table S2 in thesupplementary material). This indicates that both phases of AMPproliferation were affected when EGFR signaling wasdownregulated. Similar results were observed when the dominant-negative form of Raf (RafDN) was induced in the AMPs (see Fig.S2E and Table S2 in the supplementary material).

In further tests we generated AMP clones defective in EGFRsignaling using the MARCM system. AMPs mutant for Egfr(Egfr[CO]) or Ras (Ras1Δc40b) did not proliferate during larvaldevelopment (Fig. 4; see Table S1 in the supplementary material).Instead of forming multiple GFP-positive clusters as in controls(Fig. 4A), these mutant clones appeared as single GFP-positive cells(Fig. 4B,C). We conclude that EGFR/RAS/MAPK signaling isrequired for AMP proliferation during both early and late larvaldevelopment.

Drosophila MAPK is activated in the AMPsTo examine whether downstream components of EGFR signalingare activated in the AMPs, we stained the larval midgut withantibodies against diphospho-extracellular signal-regulated kinase(dpERK; Rolled – FlyBase), the level of which is a directmeasurement of the activated form of Drosophila MAPK (Gabay etal., 1997). dpERK staining was indeed detected in the AMP clusters,but not in the larval gut epithelial cells (Fig. 5A-A�), indicatingactivation of MAPK in these cells. This result is consistent with ourgenetic results and supports the notion that EGFR signalingregulates AMP proliferation through activating the MAPK pathway.

487RESEARCH ARTICLEDrosophila AMP development

Fig. 3. Activated Ras (RasV12) stimulates AMP proliferation. UAS-transgenes were induced in the Drosophila AMPs using the esgGal4ts

system. Larvae were shifted to 29°C at the indicated times anddissected at 96 hours AED. (A) GFP (24-96 hours AED, control).(B) RasV12 (72-96 hours AED). (C) RasV12 (48-96 hours AED). (D) RasV12

(24-96 hours AED). (E,F) Cross-sections of posterior midguts fromwandering L3 larvae expressing ectopic GFP (E, wild type, WT) or RasV12

(F) throughout larval development (24-120 hours AED). The controlAMP clusters are basally localized in the epithelium (E, arrows).PM, peritrophic membrane. The samples in E and F were stained withToluidine Blue.

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Next, we examined the expression patterns of several EGFRligands in the larval midgut. We identified a Gal4 enhancer trapin spi (NP0261, see Materials and methods) that drove UAS-GFPexpression specifically in the AMPs (Fig. 5B-B�). RNA in situhybridization confirmed that spi was specifically expressed in theAMP clusters (Fig. 5D,D�). Krn was also specifically expressedin the AMPs as shown by RNA in situ hybridization (Fig. 5E,E�).

Multiple EGFR ligands are involved in AMPproliferationTo investigate which EGFR ligands regulate the proliferation of theAMPs, we expressed each of the four known EGFR activatingligands (gurken, spitz, Keren and vein) in the AMPs using theesgGal4ts system. Induction of activated gurken (grkΔTC), a strongEGFR ligand, the function of which is believed to be exclusively infemale oogenesis (Nilson and Schupbach, 1999), did not affect theproliferation of the AMPs (see Table S2 in the supplementarymaterial). However, induction of activated (secreted) spitz or Keren(sSpi or sKrn), two other strong EGFR activating ligands (Reich andShilo, 2002; Schweitzer et al., 1995), promoted extensiveoverproliferation of the AMPs (Fig. 6A-C; see Table S2 in thesupplementary material). Furthermore, induction of wild-type Krn,which requires cleavage by rhomboid family proteases to becomefully active, similarly promoted AMP proliferation (Fig. 6D-D�; seeTable S2 in the supplementary material). In addition, induction ofsSpi or sKrn limited the dispersal of the AMPs, thus reducing thenumber of the clusters in the wandering L3 larval midguts (Fig. 6E).Induction of the weak EGFR ligand vein (vn) (Schnepp et al., 1998)with esgGal4ts, however, had little effect on AMP proliferation (seeTable S2 in the supplementary material).

To determine whether spi or Krn are required for AMPproliferation, we downregulated the levels of these EGFR ligands inthe AMPs by RNAi. Ectopic expression of UAS-RNAi directed at spiand/or Krn using esgGal4 had no effect on AMP proliferation (seeTable S2 in the supplementary material). Consistent with this, theproliferation of the AMPs in Krn27-3-4 (null allele) mutant larvae wasnormal (see Table S1 in the supplementary material). The same wasalso found for spiA14 mutant AMP clones generated in a Krn27-3-4

homozygous mutant background (see Table S1 in the supplementarymaterial). The proliferation of mutant AMPs lacking rhomboid(rhodel1, null allele) or spi (spiA14, null allele) function, generatedusing the MARCM system, was also normal (see Table S1 in thesupplementary material). We examined lacZ expression from tworholacZ reporters in the larval midgut (rhoAA69 and rhoX81) and foundthat neither were expressed in the AMPs. Since the Drosophilagenome encodes multiple rhomboid-like genes, we also generatedMARCM clones in the mutant background of rho-2 (stet871) andrho-3 (ru1). These mutant clones also contained normal numbers ofAMP clusters (see Table S1 in the supplementary material). Theseresults suggest that either multiple, redundant rhomboid-like genesare utilized in the AMPs, or (less likely) that rhomboid-like functionis dispensable in the larval midgut. Furthermore, we conclude thatspi and Krn are likely to be dispensable for AMP proliferation (seeDiscussion).

Surprisingly, in several vn mutants (vnP1749/P1749, vnγ7/γ7 andvnP1749/γ7), few AMP clusters were found in the late larval midgut (Fig.7B,C), whereas the AMPs in wild-type controls formed many largeclusters (Fig. 7A). This suggests that vn is required for normal AMPdevelopment. To further study the function of vn in AMPdevelopment, we carried out lineage analysis of the AMPs in the vnmutant animals. We induced GFP-marked AMP clones in first instarlarvae using the Flp/Gal4 system. Compared with control midguts,which contained on average 15.2 marked AMP clusters per clone(n=50 clones) (see Fig. S3A-A� in the supplementary material), weconsistently observed only a single GFP-positive AMP cluster in themidguts of weak vn mutants (vnγ7/P1749; animals of this genotype arenot developmentally delayed during larval development and most dieas pharate adults) (see Fig. S3B-B� in the supplementary material).Furthermore, we counted the number of esg-positive cells (marked byesglacZ) in newly hatched larval midguts. Control larval midguts

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Fig. 4. EGFR signaling mutant AMPs fail to proliferate. Egfr–/– orRas–/– Drosophila AMP clones were generated using the MARCMsystem, which positively marks mutant cells with GFP expression.(A) FRT42D only (control). (B) FRT42D Egfr[CO]. (C) FRT82B RasΔc40b. Theboxed regions in A-C are shown to the right as GFP (A�-C�), DNA(A�-C�) and merged (A�-C�) images. Unlike in the control (A-A�), whereGFP-positive clones form multiple clusters, clones of Egfr[CO] andRasΔc40b AMPs (B-B�; C-C�) appear as single GFP-positive cells.Arrowheads indicate the positions of Egfr–/– or Ras–/– AMPs. The asteriskin C indicates one larval EC with non-specific GFP expression from theMARCM system (FRT82B).

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(vnP1749/+) contained on average 121 AMPs per gut, whereas therewere on average 137 AMPs per midgut in vnγ7/P1749 mutants (tenmidguts for each genotype were scored). This indicates that thereduction in the number of AMP clusters in the late larval midgut ofvnγ7/P1749 mutants was not due to the production of fewer AMPsduring embryogenesis. Taken together, these results suggest that theproliferation of AMPs during the early larval stages is completelyinhibited in vn mutants. However, the size of the few remaining AMPclusters in the vnγ7/P1749 mutant midguts was relatively normal (seeFig. S3B-B� in the supplementary material), suggesting that the latephase of AMP proliferation is largely unaffected in vn mutants. Wespeculate that the reason vn becomes dispensable for AMPproliferation during late larval development is that Krn and spiexpression in the AMPs supplies a redundant function.

Interestingly, we found that vn is specifically expressed in VMcells throughout larval development, as revealed by the expressionof a well-characterized lacZ enhancer-trap insertion, vnlacZP1749

(Fig. 5C-C�) (Kiger et al., 2000). The Drosophila midgut VMcomprises an outer layer of 21 longitudinal muscle strips and aninner layer of circular muscle that forms four distinctive rows(Klapper, 2000), as revealed by a muscle-specific driver,howGal424B, which drives UAS-GFP expression in both types ofVM cells (Fig. 5C�) (Brand and Perrimon, 1993). vnlacZ expressionwas stronger in the inner, circular VM cells than in the outer,longitudinal muscle (Fig. 5C).

To test the importance of Vn signaling from VM, we specificallydepleted vn from VM cells by expressing UAS-Vn RNAi using aninducible muscle-specific driver, howGal4ts. Induction of vn RNAi

throughout larval development (24-120 hours AED) resulted in latelarval midguts with very few AMP clusters, as in vn mutants (Fig.7D-D�). However, induction of vn RNAi starting at early third instar(72 hours AED) had no effect on the AMPs (Fig. 7E-E�).Furthermore, induction of UAS-Vn RNAi in the AMPs or in thelarval ECs using the esgGal4ts or MyoIAGal4ts (MyoIA is alsoknown as Myo31DF – FlyBase) drivers had no effect on AMPproliferation (see Fig. S4A,B in the supplementary material),suggesting that the principal source of Vn is VM. This wasconfirmed by quantitative real-time PCR showing that induction ofUAS-Vn RNAi in VM significantly reduced vn mRNA levels inwhole midguts, whereas induction of vn RNAi in ECs or AMPs didnot (Fig. 7G). In further tests, we attempted to rescue the AMPphenotype of vnP1749 mutants by expressing UAS-Vn in AMPs, ECsor VM, using the esgGal4ts, MyoIAGal4ts or howGal4ts drivers.Induction of UAS-Vn in the AMPs or VM completely rescued thephenotype of vnP1749 mutants (Fig. 7F-F�; see Fig. S5A-A� in thesupplementary material). Induction of vn in the larval ECs, whichconstitute the bulk of the midgut mass, not only rescued theproliferative defects of the AMPs, but also caused ectopic AMPproliferation (see Fig. S5B-B� in the supplementary material). Weconclude that Vn, expressed in VM, is the principal mitogen forAMPs during early larval development. Later, autocrine Spi and Krnmight complement this function.

This scenario, in which VM-derived Vn activates EGFR signalingin AMPs, is reminiscent of the role of Vn in muscle/tendondevelopment during embryogenesis. In this case, muscle-derived Vnis specifically concentrated on tendon cells and activates EGFR

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Fig. 5. Expression and activity of the EGFRligands spitz, Keren and vein in the larvalmidgut. (A-A�)MAPK activity (dpERK staining) inthe midgut of late third instar Drosophila larvae.(B-B�) The expression of UAS-GFP driven byspiGal4NP0261 in the midgut of L3 wandering larvae.(C-C�)vnlacZ reporter expression pattern in themidgut. Large arrows indicate the circular visceralmuscle cells, which form four distinct rows (two areshown). Small arrows indicate the longitudinalvisceral muscle cells. (D,D�)Krn RNA in situhybridization in L3 wandering larval midgut.(E,E�) spi RNA in situ hybridization in L3 wanderinglarval midgut. Arrowheads indicate the positions ofthe AMP clusters in all panels.

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there (Strumpf and Volk, 1998). The concentration of Vn is highlydependent upon the activity of the short stop (shot, also calledkakapo) gene in the tendon cells (Strumpf and Volk, 1998). Wetested whether shot is also required for VM-derived Vn to activate

EGFR signaling in the AMPs by quantifying AMP clusters in shotmutant MARCM clones. These clones all contained normalnumbers of AMP clusters (see Table S1 in the supplementarymaterial), and thus the role of shot in transducing the Vn signal isuncertain.

DISCUSSIONDrosophila AMPs undergo extensive proliferationduring larval developmentDrosophila AMPs were previously thought to be relativelyquiescent during larval development, dividing just once or twice,and not initiating rapid proliferation until the onset ofmetamorphosis (Bodenstein, 1994). This is the case for severalother larval progenitor/imaginal cell types, such as the abdominalhistoblasts and cells in the salivary gland, foregut and hindgutimaginal rings (Bodenstein, 1994). More recent studies havesuggested that AMP proliferation might precede the onset ofmetamorphosis (Hall and Thummel, 1998; Jiang et al., 1997; Li andWhite, 2003). However, these studies did not report the extensiveproliferation of the AMPs that we describe here, and failed torecognize the early larval proliferative phase when the AMPsdivide and disperse (Figs 1 and 8). The extensive proliferation ofthe AMPs is similar to that of the larval imaginal disc cells, whichalso proliferate throughout larval development, dividing about tentimes.

Lineage analysis revealed that the proliferation of the DrosophilaAMPs occurs in two distinct phases (Fig. 8). In early larvae, theAMPs divide and disperse throughout the midgut to form individualislets. During later larval development, the AMPs continue to dividebut do so within these islets, forming large cell clusters. Wespeculate that in the early larva, secretion of Vn from the midgutvisceral muscle (VM) cells results in low-level activation of EGFRsignaling in the AMPs, which is sufficient for their proliferation andmight also promote their dispersal. We did not observe anyproliferation defects in AMPs defective in shot function, suggestingthat the mechanism of EGFR activation used by tendon cells duringmuscle/tendon development is probably not the same as in the larvalmidgut. Specifically, it is unlikely that the Shot-mediatedconcentration of Vn on AMPs activates EGFR signaling in theAMPs during early larval development. Consistent with this, weonly observed dpERK staining in AMP clusters (Fig. 5A-A�) andnot in the isolated AMPs present at early larval stages (24-72 hoursAED; data not shown).

The mechanisms that regulate the transition between these twoproliferation phases remain unclear. We observed fewer AMPclusters when sSpi, sKrn, λTOP (activated Egfr) or RasV12 wereinduced in the AMPs starting from early larval stages (Fig. 6E; seeTable S2 in the supplementary material), suggesting that EGFRsignaling, in addition to its crucial role as an AMP mitogen, mightalso play a role in AMP cluster formation. In the late larval midgut(96-120 hours AED), high-level EGFR activation, resulting fromexpression of spi and Krn in the AMPs themselves, might not onlypromote AMP proliferation, but might also suppress AMPdispersal and thus promote formation of the AMP clusters. Howthe timing and location of Spi- or Krn-mediated EGFR activationare regulated during larval development is also unclear. We note,however, that the pro-ligand form of Krn acted similarly to sKrn(Fig. 6), and that we failed to uncover any functions for the Rho-like gene products that regulate Spi and Krn function byproteolytic cleavage in other tissues (see Tables S1 and S2 in thesupplementary material). This suggests that the localizedexpression of these ligands in the AMP clusters might be the

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Fig. 6. Expression of sSpi, Krn or sKrn in the AMPs induces theirproliferation. The ligands were induced in the Drosophila AMPs usingthe esgGal4ts system starting at 24 hours AED, and larvae weredissected at 96 hours AED. (A,A�)GFP (control). (B,B�)Activated(secreted) Spi (sSpi). (C,C�)Activated (secreted) Krn (sKrn). (D,D�)Krn.(A-D) GFP marks the AMP clusters. (A�-D�) Merged images of GFP(green) and DNA (DAPI, blue). (E) The ectopic expression of strong EGFligands in the AMPs dramatically reduces the total number of AMPclusters in the midgut. WT, wild-type.

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critical parameter that controls their effects. Consistent with this,Rho-independent cleavage and function of Krn have beendocumented (Reich and Shilo, 2002).

In the developing Drosophila wing, EGFR/RAS/MAPKsignaling promotes the expression and controls the localization ofthe cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin)(O’Keefe et al., 2007). RasV12-expressing clones generated in thewing imaginal disc are round (Prober and Edgar, 2002), much likethe AMP clusters described here, owing to increased adhesivejunctions. In developing Drosophila trachea, EGFR activityupregulates shg expression to maintain epithelial integrity in theelongating tracheal tubes (Cela and Llimargas, 2006). In the eye,EGFR activity leads to increased levels of Shg and adhesionbetween photoreceptors (Brown et al., 2006; Mirkovic andMlodzik, 2006). Given these precedents, it seems reasonable tosuggest that high-level EGFR activity in the AMP islets upregulatesShg and promotes the homotypic adhesion of the AMPs.Alternatively, changes in the differentiated cells of the midgutepithelium might promote AMP clustering. In either case, thedispersal of early AMPs and subsequent formation of late AMPclusters facilitate the formation of the adult midgut epitheliumduring metamorphosis.

AMPs give rise to adult intestinal stem cellsduring metamorphosisOur study confirms previous reports that Drosophila AMPs replacelarval midgut epithelial cells to form the adult midgut epitheliumduring metamorphosis (Figs 1 and 8) (Bender et al., 1997; Jiang et al.,1997; Li and White, 2003). Furthermore, we show that the majorityof AMPs lose esgGal4-driven GFP expression as they differentiate toform the new adult midgut epithelium (Fig. 1F-J). These cells lackedProspero, which marks enteroendocrine cells in both the larval andadult midgut (Micchelli and Perrimon, 2006; Ohlstein and Spradling,2006). They went through several rounds of endoreplication duringlate pupal development (not shown), and thus probably alldifferentiated into adult enterocytes (ECs). During earlymetamorphosis, some cells in the new midgut epithelium remainedsmall and diploid and maintained strong esgGal4 expression (Fig.1I,J; Fig. 8). For several reasons, we believe that these esg-positivecells are the future adult intestinal stem cells (ISCs). First, esgGal4expression marks AMPs, including adult ISCs and enteroblasts(Micchelli and Perrimon, 2006). Second, mitoses in the adult midgutare only observed in ISCs (Micchelli and Perrimon, 2006; Ohlsteinand Spradling, 2006), and we observed mitoses only in the esg-positive cells during metamorphosis (see Fig. S1 in the supplementary

491RESEARCH ARTICLEDrosophila AMP development

Fig. 7. Vein is required for AMP proliferation.(A-C) Posterior midguts from white prepupa (0 hours APF) ofwild-type (WT) Drosophila contain multiple AMP clusters (A),which are missing from the midguts of vn mutants (B, vnP1749;C, vnγ7). Midguts are outlined with dashed lines.(D-E�) Posterior midguts from wandering L3 larvae in whichvn was specifically knocked down in the visceral muscle cellsthroughout larval development (24-120 hours AED, D) or onlyduring late larval development (72-120 hours AED, E). Mostof the remaining small cells in B-D are visceral muscle cells.Arrows in D point to the few AMP clusters in the midgut.(F,F�) Induction of UAS-Vn expression throughout larvaldevelopment (24-120 hours AED) using the muscle-specificdriver howGal4ts rescued the vn mutant phenotype. D�-F�show merged images of DNA (DAPI, blue) and howGal4ts-driven GFP expression (green) in the visceral muscle andtrachea (asterisks in D�,F�) cells. (G) Knockdown of vn mRNAin the midgut by vn RNAi. Relative levels of vn mRNA in thelarval midgut were quantified by qRT-PCR. Only UAS-Vn RNAiexpression driven by the muscle-specific Gal4 driver,howGal4ts, knocked down vn significantly in the larvalmidgut.

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material). Third, esg-positive cells migrated to the basal side of themidgut epithelium (Fig. 1J), the location of adult ISCs (Micchelli andPerrimon, 2006; Ohlstein and Spradling, 2006). Fourth, AMP clonesgenerated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF)(see Fig. S1C-C� in the supplementary material), but when such cloneswere scored in newly eclosed adults, they contained large numbers ofECs, as well as cells positive for the enteroendocrine marker Prosperoand the ISC marker Delta (Fig. 2D,E). This suggests that a smallfraction of AMPs differentiate into adult ISCs. However, esg-positivecells in the new pupal midgut lacked Delta expression until eclosion(Fig. 2E-E�; data not shown), suggesting that they are probably notmature adult ISCs.

How a small fraction of AMPs are selected to become adult ISCsin the newly formed pupal midgut epithelium is not known. Onepossibility is that the adult ISCs are determined during larvaldevelopment, long before the formation of the adult midgut. Anotheris that they are specified during early metamorphosis. We prefer thissecond hypothesis for several reasons. First, in our lineage analysis,we found that all AMP clones induced during early larval stagesformed multiple clusters (Fig. 2A-A�; see Fig. S3A-A� in thesupplementary material). This suggests that there are no quiescentAMPs in the larval midgut. Second, when AMP clones were inducedat mid-third instar, the mosaic clusters always contained multipleGFP-positive cells, suggesting that all AMPs in the mid-third instarmidgut remain equally proliferative (Fig. 2B-B�). Third, duringlarval development, we never observed differentiation of the AMPs,as judged by their ploidy (diploid) and lack of expression of theenteroendocrine marker Prospero (not shown). Fourth, all AMPsappeared to express esgGal4 throughout larval development. Giventhe crucial role that Notch signaling plays in regulating AMPsduring embryonic midgut development (Hartenstein et al., 1992) andISCs in adult midgut homeostasis (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007), weexpect that Notch might also function to specify adult ISCs duringmetamorphosis.

Implications for EGFR/RAS signaling in insectmidgut developmentEGFR signaling is both required and sufficient to promote AMPproliferation (Figs 3, 4, 6 and 7; see Fig. S2 in the supplementarymaterial). Hyperactivation of EGFR signaling, such as by expressionof activated Ras (RasV12), promoted massive AMP overproliferationand generated hyperplastic midguts that were clearly dysfunctional(Fig. 3F). On the other hand, inhibiting EGFR/RAS/MAPK

signaling dramatically reduced AMP proliferation (Fig. 4; see Fig.S2 in the supplementary material). Furthermore, the ability of EGFRsignaling to induce ectopic AMP proliferation is almost unique.With the exception of larval hemocytes (Zettervall et al., 2004),activated EGFR signaling does not promote cell proliferation in theimaginal discs, salivary gland imaginal rings, abdominal histoblasts,foregut and hindgut imaginal rings. This suggests that the regulationof AMP proliferation is different from that in other imaginal cells.

Regulation of AMP proliferation by non-epithelialmuscle cellsDespite the obvious differences between adult ISCs and theirlarval progenitors, the AMPs, there are also similarities. First,when the new adult midgut epithelium forms, larval AMPs giverise to the new adult midgut including the adult ISCs. Manygenes, such as esg, that are specifically expressed in the larvalAMPs are also expressed in the adult ISCs (our unpublished data).Second, the structure of the midgut epithelium with basal AMPsor ISCs is similar in larval and adult stages. Third, vn expressionin larval VM persists in the adult midgut (our unpublished data),suggesting that Vn from the adult VM might also regulate theISCs.

In two Drosophila stem cell models, the testis and ovary, stemcells reside in special niches comprising other supporting cell types.These niches maintain the stem cells and provide them withproliferative cues (Ohlstein et al., 2004). For example, in the testis,germ stem cells attach to the niche that comprises cap cells. The capcells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp],which maintain the stem cells and induce their proliferation.Whether Drosophila ISCs utilize supporting cells that constitute aniche remains unclear. Here we show that multiple EGFR ligandsare involved in the regulation of Drosophila AMP proliferation.During early larval development, the midgut VM expresses theEGFR ligand vn (Fig. 5C-C�), which is required for AMPproliferation (Fig. 7; see Fig. S3B-B� in the supplementarymaterial). Thus, the early AMPs might be considered to require aniche comprising non-epithelial VM. Later in larval development,however, the AMPs express two other EGFR ligands, spi and Krn(Fig. 5E,F), which are capable of autonomously promoting theirproliferation (Fig. 6) and may render vn dispensable (Fig. 7E,E�; seeFig. S3B-B� in the supplementary material). We found, however,that depleting spi and Krn in the AMPs did not affect AMPproliferation, suggesting that vn or another trigger ofEGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae.

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Fig. 8. Postembryonic development of theDrosophila midgut epithelium. AMPs (green)proliferate in two phases and several EGFR ligandsare involved in each phase. Also note thespecification of future adult intestinal stem cellsduring early metamorphosis and the reappearance ofenteroendocrine cells (red) at a late stage ofmetamorphosis (72 hours APF). See text for details.

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We thank Amanda Simcox, Matthew Freeman, Jocelyn McDonald, Ryu Ueda,Celeste Berg, Laura Johnston, Andrew Dingwall, Margaret Fuller and the NIG(Japan), VDRC (Vienna) and Bloomington (USA) Stock Centers for providingflies; the FHCRC EM Laboratory for preparing larval midgut sections; theMoen’s lab for their help with confocal imaging; two anonymous reviewers fortheir helpful comments; and members of Edgar lab for their support, especiallyDr Tao Wang, Dr Parthive Patel and Aida de la Cruz for critical reading of themanuscript. This work was supported by pilot funds from the UW/FHCRCCancer Consortium and NIH grant R01 GM51186 to B.A.E. Deposited in PMCfor release after 12 months.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/3/483/DC1

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