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write a Critique and a summary in a separate pages Article Regulation of Drosophila intestinal stem cell maintenance and differentiation by the transcription factor Escargot Mariano A Loza-Coll1,2, Tony D Southall3,†, Sharsti L Sandall1, Andrea H Brand3 & D Leanne Jones1,2,4,* Abstract Tissue stem cells divide to self-renew and generate differentiated cells to maintain homeostasis. Although influenced by both intrin- sic and extrinsic factors, the genetic mechanisms coordinating the decision between self-renewal and initiation of differentiation remain poorly understood. The escargot (esg) gene encodes a tran- scription factor that is expressed in stem cells in multiple tissues in Drosophila melanogaster, including intestinal stem cells (ISCs). Here, we demonstrate that Esg plays a pivotal role in intestinal homeostasis, maintaining the stem cell pool while influencing fate decisions through modulation of Notch activity. Loss of esg induced ISC differentiation, a decline in Notch activity in daughter enteroblasts (EB), and an increase in differentiated enteroendo- crine (EE) cells. Amun, an inhibitor of Notch in other systems, was identified as a target of Esg in the intestine. Decreased expression of esg resulted in upregulation of Amun, while downregulation of Amun rescued the ectopic EE cell phenotype resulting from loss of esg. Thus, our findings provide a framework for further compara- tive studies addressing the conserved roles of Snail factors in coor- dinating self-renewal and differentiation of stem cells across tissues and species. Keywords Amun; Drosophila; Escargot; Notch; stem cell Subject Categories Development & Differentiation; Stem Cells; Transcription DOI 10.15252/embj.201489050 | Received 21 May 2014 | Revised 20 October 2014 | Accepted 27 October 2014 | Published online 28 November 2014 The EMBO Journal (2014) 33: 2983–2996 See also: J Korzelius et al (December 2014) Introduction During homeostasis, tissue stem cells maintain the stem cell popula- tion through self-renewal and give rise to differentiating progeny to replace cells lost to normal turnover of the tissue (Biteau et al, 2011; Simons & Clevers, 2011; Wang & Jones, 2011). In response to acute and chronic stress (infection, wounding, aging, metabolic challenges), tissue stem cells can undergo dynamic waves of symmetric self-renewing or differentiating divisions to quickly prop- agate and replace damaged tissue (Morrison & Kimble, 2006; Egger et al, 2010; O’Brien et al, 2011; Piccin & Morshead, 2011; Simons & Clevers, 2011). While significant progress has been made in the identification, isolation and manipulation of tissue stem cells in organisms ranging from plants to vertebrates (Amatruda & Zon, 1999; Gentile et al, 2011; Takashima et al, 2013), our understanding of conserved mechanisms that regulate the choice between self- renewal and the onset of differentiation in vivo is lacking. In this regard, model organisms such as Caenorhabditis elegans and Drosophila melanogaster have been instrumental for the character- ization of basic regulatory mechanisms in stem cells, such as the role of asymmetric divisions (Yamashita et al, 2003; Wu et al, 2008; Egger et al, 2010; Inaba & Yamashita, 2012) and the interaction between stem cells and their niche (Wong et al, 2005; Spradling et al, 2008; Losick et al, 2011; Resende & Jones, 2012). The identification and characterization of stem cells in the posterior midgut of adult flies (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006) has revealed numerous regulatory mechanisms conserved between flies and vertebrates (Biteau et al, 2011; Jiang & Edgar, 2012). The Drosophila midgut epithelium is composed of intestinal stem cells (ISCs), enteroblasts (EBs), secretory enteroen- docrine (EE) cells and absorptive enterocytes (ECs) (Fig 1A). Through cell division, ISCs self-renew to maintain the ISC pool and generate progenitor cells, which adopt either an EE or an EC fate. In addition, ISCs can divide symmetrically to generate either two daughter ISCs or two cells that will differentiate (O’Brien et al, 2011; Goulas et al, 2012; de Navascues et al, 2012). Indeed, it has been proposed that intestinal homeostasis in flies is maintained through a neutral drift between all possible mitotic outcomes (de Navascues et al, 2012). ISCs express the ligand Delta (Dl), which activates Notch (N) signaling in adjacent EBs to promote differentiation and influence cell fate decisions: a weak N signal specifies EE fate, whereas 1 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA 2 Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA 3 The Gurdon Institute, University of Cambridge, Cambridge, UK 4 Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA, USA *Corresponding author. Tel: +1 310 206 7066; E-mail: [email protected] †Present address: Department of Life Sciences, Imperial College London, London, UK ª 2014 The Authors The EMBO Journal Vol 33 | No 24 | 2014 2983 http://dx.doi.org/10.15252/embj.201489072 stronger N signaling generates ECs (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006, 2007). Accordingly, strong loss-of-function mutations in the N pathway cause an accumulation of ISC-like cells, due to lack of EB differentiation, whereas weaker loss-of-function mutations of Notch generate clusters of ISC-like cells and EEs, due to a combination of impaired EB differentiation and a bias toward the EE fate. In contrast, ectopic activation of N in ISCs results in precocious differentiation, with a bias toward the EC fate (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006, 2007). While the regulation of the ISC lineage by the Notch pathway and its down- stream effectors has been well established previously (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006; Bardin et al, 2010; Perdigoto et al, 2011), little is known about upstream mechanisms that control the levels of Notch activity in this system. The expression of esg reporter transgenes has been used to mark ISCs and EBs since their initial characterization (Micchelli & Perri- mon, 2006). Subsequently, the restricted expression of endogenous esg mRNA in ISC/EB nests was confirmed by fluorescence in situ hybridization in combination with immunofluorescence staining (FISH/IF) (Fig 1B; Toledano et al, 2012). To date, however, whether esg plays any specific role in the regulation of ISCs remains unknown. Esg is a member of the Snail family of transcription factors that act primarily through competition with transcriptional activators for access to a consensus-binding site, the E-box, within the promoter region of target genes (Hemavathy et al, 2000; Nieto, 2002; Barrallo- Gimeno & Nieto, 2005). The Snail factors were first characterized in Drosophila and are conserved from mollusks to humans (Nieto, 2002). In addition to expression in ISCs, Esg is expressed in germ- line stem cells (GSCs) and cyst stem cells (CySCs) of the testis (Kiger et al, 2000; Voog et al, 2014) and, during development, in neural stem cells and imaginal disks (Hayashi et al, 1993; Ashraf et al, 1999; Cai et al, 2001). Moreover, our previous work demonstrated that Esg is required for the maintenance of CySCs and hub cells, a critical component of the stem cell niche in the adult testis (Voog et al, 2014). Given the restricted expression of esg in ISC/EBs of the Drosoph- ila intestine, we sought to characterize the function of Esg in these cells. Here, we demonstrate that Esg is required for maintenance of ISCs and an important regulator of Notch signaling within EBs. Furthermore, DNA binding analysis by DamID identified Amun, a previously characterized negative regulator of Notch signaling (Abdelilah-Seyfried et al, 2000; Shalaby et al, 2009), as a putative target of Esg in the gut. Accordingly, Esg knockdown in ISC/EBs caused an upregulation in Amun expression in these cells. Further- more, abrogating the increase in Amun rescued the reduction in Notch activity and accumulation of EE cells caused by loss of Esg. Based on our data, we conclude that Esg positively modulates Notch signaling within EBs through repression of Amun, influencing the decision between EC and EE fates. Therefore, we propose that Esg plays a pivotal role in intestinal homeostasis, simultaneously promoting stem cell maintenance and regulating the differentiation of EBs. Results Loss of Escargot function in ISC/EBs leads to loss of ISCs and a bias toward the enteroendocrine cell fate Most esg alleles result in lethality during development when homo- zygous; however, the shutoff (shof) allele of esg is a homozygous viable mutation in the esg locus, which permits investigation of adult phenotypes (Voog et al, 2014). FISH/IF analysis revealed that esgshof homozygotes express normal levels of esg mRNA in ISC/EBs (Supplementary Fig S1A), and intestines from these flies appeared normal. Therefore, in order to probe the role of Esg in the intestine, FRT-mediated recombination was used to generate ISCs homozy- gous mutant for a null allele of esg, esgG66 (Whiteley et al, 1992; Lee & Luo, 1999; Voog et al, 2014). In this experiment, a heat shock- induced recombination generates esg mutant cells that become permanently labeled by expression of GFP. Progeny derived from marked ISCs are similarly marked, permitting characterization of cells derived from esg mutant ISCs (or that of corresponding wild- type counterparts, in control animals). Clones of esgG66 mutant cells did not appear grossly different from wild-type clones at early time ▸Figure 1. Loss of Escargot induces ISC loss and a bias toward the enteroendocrine cell fate.A Drosophila posterior midgut epithelium. Schematic representation of cell types and their lineage relationships (see text for details; Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006). Intestinal stem cells (ISCs) and enteroblasts (EBs) can be usually identified by an esg-GFP reporter, or expression of GFP under control of ISC/EB-specific drivers. ISCs express Delta (Dl, which results in a characteristic punctate staining of ISCs), which activates Notch in EBs (revealed by b-GAL staining of flies that carry a Su(H)-lacZ reporter of Notch activity (Bray & Furriols, 2001)). Enteroendocrine cells (EE) are identified by nuclear Prospero (Pros) staining, whereas enterocytes (ECs) can be distinguished based on their large polyploidy nuclei (as revealed by DAPI staining of DNA). B esg mRNA is restricted to ISC/EB cells. FISH/IF staining for esg mRNA (red, gray) and GFP protein (green) in midguts of 3- to 5-day-old adults carrying an ISC/EB- specific reporter (“esg > GFP” = esg-Gal4, UAS-GFP). C Clonal analysis of esg mutant ISCs. Representative images of wild-type (control) and esg mutant (esgG66) MARCM clones stained as indicated with DAPI, Pros and GFP, 4 or 10 days after heat shock (dphs). esgG66 mutant clones are smaller and contain EE cells more frequently than controls (arrows). D, E Loss of esg results in loss of ISCs and an increase in EE cells. A CellProfiler analysis of images as those in (C) confirmed that esgG66 mutant clones are significantly enriched for EE cells (D) and have significantly less cells (E) than their control counterparts (***P < 0.001 and **P < 0.01, Kruskal–Wallis/Dunn test). F, G Phenotypes induced by RNAi-mediated depletion of esg in ISC/EBs. (F) RNAi-mediated knockdown of Esg in ISC/EBs caused an accumulation of EE cells and a noticeable change in the morphology and size of some ISC/EBs (arrows in bottom panel; e.g. compare the large GFP+ cell identified by the rightmost arrow to its two smaller neighbors). Midguts from adults of the indicated genotypes were stained with DAPI (all nuclei), GFP (ISC/EB) and Pros (EE cells) following a 6-day incubation at 25°C on 10 lg/ml RU486 or EtOH-containing food (as indicated). (G) Images as those in (F) were processed with CellProfiler to quantify the relative proportion of EE cells in the indicated genotypes/treatments (see Materials and Methods for details). Each data point is an average proportion calculated from four independent images per midgut, and the bars are the geometric mean � SEM of those averages. *** denotes a significant enrichment of EE cells following Esg knockdown in ISC/EBs compared to either control group (P < 0.001, Kruskal–Wallis/Dunn test). Data information: Scale bars = 20 lm. The EMBO Journal Vol 33 | No 24 | 2014 ª 2014 The Authors The EMBO Journal Regulation of intestinal stem cells by Escargot Mariano A Loza-Coll et al 2984 A B C F D E G Figure 1. ª 2014 The Authors The EMBO Journal Vol 33 | No 24 | 2014 Mariano A Loza-Coll et al Regulation of intestinal stem cells by Escargot The EMBO Journal 2985 points (Fig 1C, 4 dphs); however, quantification of Prospero- expressing (Pros+) cells within esgG66 clones revealed a significant enrichment of EE cells (Fig 1D). At later time points, esgG66 clones were significantly smaller than control clones (Fig 1C and E, 10 dphs) and remained significantly enriched for EE cells (Fig 1D). We used CellProfiler (Carpenter et al, 2006; Kamentsky et al, 2011) to automatically classify and quantify cells within GFP+ esgG66 and control clones (Supplementary Fig S1B and Supplemen- tary Table S1; see Materials and Methods for details). Our analysis showed a higher prevalence of multicellular esgG66 clones that contained only differentiated cells, consistent with a role for Esg in ISC maintenance (polyploid ECs, EE cells or combinations thereof, examples are shown in Supplementary Fig S1C, insets iv and v). The proportion of esgG66 that did not contain ISCs or EBs was approximately double that of wild-type counterparts, both at 4 and 10 dphs (Supplementary Fig S1D). In addition, esgG66 clones lacking ISC/EBs had a significantly larger proportion of EE cells compared to controls (Supplementary Fig S1E). Of note, the frequency of wild- type clones that lost the ISC at 4 dphs (12.5%) is in close agreement with previously reported rates of symmetric/differentiating ISC divi- sions (O’Brien et al, 2011; Goulas et al, 2012; de Navascues et al, 2012). Particularly evident among esgG66 GFP+ clones were instances of Pros+ doublets (Supplementary Fig S1C, inset v), which were only rarely observed in control clones (3.13% of esgG66 clones with more than one cell vs. 0.26% of control clones, Supplementary Table S1). To confirm these findings, we used the GAL4-UAS system to induce RNAi-mediated knockdown of Esg expression. Specifically, a UAS-esgRNAi construct (referred to as esgRNAi hereafter) was expressed under control of a drug-inducible GAL4 driver, 5961GS, whose expression pattern recapitulates esg expression in the diges- tive tract (Biteau et al, 2010; Mathur et al, 2010). Esg knockdown caused an alteration in the morphology of ISC/EBs, some of which appeared larger and had an overall morphology reminiscent of ECs (arrows in Fig 1F). In addition, a striking accumulation of Pros+ EE cells was observed (Fig 1F and G), with the total proportion doubling after 6 days of esgRNAi induction. Similar results were obtained with an independent UAS-esgRNAi line (Supplementary Fig S1F) and with another inducible and considerably stronger ISC/ EB-specific GAL4 driver (esg-Gal4, tub-Gal80ts, referred to as “esgts” hereafter) (Supplementary Fig S3C and D). Taken together, these data indicate that loss of esg function in ISC/EBs leads to a progressive loss of ISCs and a shift in differentiation toward the EE lineage. To determine in which cell type Esg is required to influence cell fates, we directed the expression of UAS-esgRNAi to ISCs or EBs. Restricted expression of UAS-esgRNAi and a UAS-2xYFP reporter to ISCs (Wang et al, 2014) revealed morphological changes in YFP+ cells consistent with the induction of ISC differentiation (Fig 2A, Supplementary Fig S2A). In addition, a significant accumulation of EE cells (Fig 2C) was also observed. Depletion of Esg exclusively in EBs, with a temperature-sensitive version of a Su(H)-Gal4 driver (referred to as Su(H)-Gal4ts, Supplementary Fig S2B) (Zeng et al, 2010), also led to an alteration in the size and morphology of EBs, including some cells that resembled polyploid ECs (Fig 2B, arrows). Interestingly, there was an even more striking and significant enrichment of EE cells (Fig 2B and D), indicating that the loss Esg function in EBs is sufficient to accelerate their differentiation and bias their fate toward the EE lineage. Loss of Escargot function in ISC/EBs correlates with reduced Notch activity in EBs Numerous reports have established a connection between activation of the Notch signaling pathway and cell fate decisions in the intes- tine (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006, 2007; Maeda et al, 2008; Bardin et al, 2010; Perdigoto et al, 2011). Notch is a transmembrane receptor that, upon binding to its ligands Delta or Serrate, undergoes a series of proteolytic cleavages leading to the cytoplasmic release of its intracellular domain (NICD or Nintra). Nintra translocates into the nucleus and binds Suppressor of Hairless, Su(H), a co-factor through which Notch activates the expression of its target genes (Koch et al, 2013). Targeted depletion of Esg in ISC/ EBs caused a noticeable reduction in the expression of a Su(H)-lacZ reporter of Notch activation in EBs (Supplementary Fig S3A), including those adjacent to ISCs that continued to express Delta (Fig 3A). These data indicate that loss of Esg leads to a decrease in N signaling between ISCs and EBs. In support of these observations, a modest induction of esgRNAi expression in flies heterozygous for a null allele of Notch (N81K1) noticeably enhanced the accumulation of EE cells, which was not obvious in either the esgRNAi or the N81K1/+ background (Fig 3B). In 17% of midguts examined (n = 18), supernumerary esg-GFP+ diploid cells were observed, along with a striking accumulation of EE cells, reminiscent of the characteristic phenotypes caused by Notch loss-of-function muta- tions (Supplementary Fig S3B). If the accumulation of EE cells upon Esg knockdown was due to a reduction in Notch activity within EBs, then ectopic activation of ▸Figure 2. Loss of Esg function causes an accumulation of EE cells and an altered EB morphology.A Phenotypes caused by depletion of esg in ISCs. Midguts of the indicated genotype were stained with DAPI (all nuclei), YFP (primarily ISCs) and Pros (EE cells) following 6 days of incubation at the indicated temperatures (control = outcross to w1118 flies). B Phenotypes caused by depletion of esg in EBs. Midguts of the indicated genotypes were stained with DAPI (all nuclei), GFP (EBs) and Pros (EE cells) following 6 days of incubation at 29°C to allow the EB-restricted expression of UAS-esgRNAi or a UAS-GFPnls (control). Notice the stronger nuclear GFP staining in control samples due to GFPnls expression. Arrows point to examples of EBs with a wider cytoplasm, dimmer GFP staining and seemingly polyploid nuclei following Esg knockdown. C EE cell accumulation induced by depletion of esg in ISCs. CellProfiler was used as in Fig 1G to quantify the relative proportions of EE cells in midguts in images as those in (A). *** and * denote a statistically significant difference in the relative proportion of EE cells in pairwise post-test comparisons indicated by the corresponding bars (P < 0.001 and P < 0.05 Kruskal–Wallis/Dunn test). D EE cell accumulation induced by depletion of esg in EBs. CellProfiler was used as in (C). *** denotes a statistically significant accumulation of EE cells following EB-specific Esg knockdown, as compared to all other samples (P < 0.001, Kruskal–Wallis/Dunn test). Data information: Scale bars = 20 lm. The EMBO Journal Vol 33 | No 24 | 2014 ª 2014 The Authors The EMBO Journal Regulation of intestinal stem cells by Escargot Mariano A Loza-Coll et al 2986 A B C D Figure 2. ª 2014 The Authors The EMBO Journal Vol 33 | No 24 | 2014 Mariano A Loza-Coll et al Regulation of intestinal stem cells by Escargot The EMBO Journal 2987 the Notch pathway through forced expression of a constitutively active Notch construct should preserve the normal balance between EEs and ECs. Expression of UAS-Nintra in ISCs and EBs with the inducible 5961GS driver resulted in strong activation of the Su(H)- lacZ reporter in both ISC and EBs (Fig 3C). Furthermore, as predicted, co-expression of Nintra and esgRNAi in ISC/EBs resulted in a suppression of ectopic EE cells (Fig 3C and D). Similar results were obtained by expressing both transgenes with the esgts driver (Supplementary Fig S3C and D). Restricted expression of Nintra and esgRNAi to EBs, using the Su(H)-Gal4ts driver, resulted in lethality, even at the restrictive temperature (18°C), likely due to leaky expression in other tissues during development. Interestingly, expression of Nintra alone in ISC/EBs led to an expected trend toward EC enrichment, which became highly significant when Nintra was expressed in combination with esgRNAi (Supplementary Fig S3E). Similarly, there was a significant accumu- lation of ECs following expression of esgRNAi alone, likely a conse- quence of ISC depletion due to loss of Esg. To address whether the proposed accumulation of EE cells reflected the non-specific, relative accumulation of ECs and EEs as a result of ISC/EB loss, we calcu- lated the relative proportion of EEs to ECs (Supplementary Fig S3F). However, this analysis confirmed the enrichment of EE cells, relative to ECs. Furthermore, the co-expression of Nintra and esgRNAi fully reversed this trend, as predicted by the model that activated Notch rescues the bias toward the EE fate in favor of EC differentiation. In summary, reduced activation of the Su(H)-lacZ reporter following esg knockdown (Fig 3A and Supplementary Fig S3A), together with the observation that Nintra is epistatic to esgRNAi (Fig 3C and D and Supplementary Fig S3D), strongly suggests that Notch functions downstream of Esg to regulate cell fate decisions in the intestine. Amun is a candidate Esg target in ISCs/EBs In order to identify mediators of Esg function in ISC/EBs, we used DamID to conduct a genome-wide in vivo mapping of Esg binding to DNA (van Steensel & Henikoff, 2000; Southall & Brand, 2009). A fusion of Esg and the bacterial DNA methylase Dam was expressed in flies, which led to the methylation of DNA surrounding Esg bind- ing sites in vivo. Genomic DNA was then extracted from midguts, and the methylated regions were labeled and hybridized to whole- genome tiling arrays (see the Supplementary Materials and Methods for further details). Putative targets of Esg in the intestine were then identified by proximity to the methylated regions identified via DamID, or “Esg binding regions” (EBRs) (Supplementary Table S2). We then used FlyMine to cross-reference genetic interactors of Notch with a list of in vivo DamID targets of Esg (Supplementary Table S2). From this approach, we identified Amun as a putative candidate (Fig 4A). Amun is a nuclear protein with a predicted DNA glycosylation domain, which has been shown to suppress Delta gain-of-function phenotypes when overexpressed in the eye and the wing (Shalaby et al, 2009) and to cause the formation of extra scutellar bristles when overexpressed in wing imaginal disks (Abdelilah-Seyfried et al, 2000). Both phenotypes are consistent with Amun acting as an inhibitor of Notch signaling. Accordingly, when we overexpressed Amun in ISC/EBs using esg-Gal4ts, we observed a down- regulation in Su(H)-lacZ expression (Fig 4B) and a corresponding enrichment of EE cells (Fig 4C). We confirmed these observations using the 5961GS driver, which led to more subtle, but reproducible, pheno- types (Supplementary Fig S4). To further explore the relationship between Esg and Amun, we tested the hypothesis that Esg knockdown would lead to upregula- tion (de-repression) of Amun. Analysis of Amun mRNA expression in whole intestines by reverse transcriptase (RT)–qPCR showed a modest but statistically significant upregulation in Amun following Esg knockdown (Supplementary Fig S5A). Because such slight upregulation could be a consequence of ISC/EBs representing only a very small proportion of the intestinal biomass, RT–qPCR measure- ments were repeated in ISC/EBs purified by FACS sorting from dissociated intestines (Dutta et al, 2013) (Supplementary Fig S5B). This complementary approach confirmed the upregulation in Amun mRNA levels following Esg knockdown (Fig 4D). We also assayed Amun expression in posterior midguts via fluorescent in situ hybrid- ization/immunofluorescence (FISH/IF) staining (Toledano et al, 2012). Although Amun mRNA was undetectable in ISC/EBs of control guts using this method, some expression could be detected within esg-GFP+ progenitor cells following RNAi-mediated depletion of esg (Supplementary Fig S5C). If Amun de-repression mediates the downregulation of Notch signaling in EBs caused by Esg depletion, then the co-expression of an AmunRNAi construct along with esgRNAi should rescue the bias in cell fates toward the EE cell lineage. We tested this prediction by co- expressing esgRNAi and AmunRNAi in EBs using the Su(H)-Gal4ts ▸Figure 3. Loss of Esg function in ISC/EBs leads to decreased Notch activity in EBs.A Esg knockdown in ISC/EBs causes reduced Notch reporter activity in EBs. Use of a Notch (N) activity reporter (Su(H)-lacZ; Bray & Furriols, 2001) reveals a noticeable reduction in N signaling within EBs, despite of seemingly unaltered Delta expression in ISCs. Midguts of the indicated genotypes were stained with DAPI (all nuclei), Delta (ISCs) and b-GAL (EBs) following 4 days of incubation in 10 lg RU486/ml or ethanol-containing food as indicated. B Epistasis analysis between Esg (esgRNAi) and Notch (N81K1). Midguts of the indicated genotypes were stained with DAPI (all nuclei), GFP (esg+ cells) and Pros (EE cells) following 6 days of incubation in 5 lg/ml RU or ethanol, as indicated. Notice that a lower concentration of RU486, resulting in a more moderate induction of esgRNAi expression, did not produce the larger and dimmer GFP+ nuclei or EE cell accumulation typically observed with higher doses of RU486. Approximately 83% (15/18) of the N81K1/+; esgRNAi guts showed a noticeable accumulation of EE cells in an otherwise normal-looking epithelium (right column, middle panel), whereas the remaining 17% showed a drastic expansion of diploid, esg+ and EE cells (right column, lower panel), reminiscent of stronger Notch loss-of-function mutations (compare with Supplementary Fig S3B). C, D Constitutively active Notch signaling rescues the EE cell enrichment phenotype caused by Esg knockdown. (C) An esgRNAi construct and a constitutively active Notch construct (Nintra) were expressed alone or in combination in ISC/EBs using the 5961GS driver. Midguts of the indicated genotypes were stained for GFP, b-GAL (EBs) and Pros (EE cells) following 7 days of incubation in ethanol or 25 lg/ml RU486 as indicated. (D) Images as those in (C) were processed with CellProfiler to quantify the relative proportion of EE cells. Midguts overexpressing the esgRNAi construct alone were the only sample that showed a significant EE enrichment relative to the corresponding EtOH control (***P < 0.001, one-way ANOVA/Bonferroni test). Data information: Scale bars = 20 lm. The EMBO Journal Vol 33 | No 24 | 2014 ª 2014 The Authors The EMBO Journal Regulation of intestinal stem cells by Escargot Mariano A Loza-Coll et al 2988 A C D B Figure 3. ª 2014 The Authors The EMBO Journal Vol 33 | No 24 | 2014 Mariano A Loza-Coll et al Regulation of intestinal stem cells by Escargot The EMBO Journal 2989 A B D C Figure 4. Amun is a candidate target of Esg. A Esg DamID profile surrounding the Amun locus. Data are displayed as custom UCSC Genome Browser tracks. Green/red bars represent the average log2 (intensity ratio) between the Esg:Dam and Dam-control samples, mapped to the genomic regions � 10 kb from Amun (see Supplementary Materials and Methods for further details). The yellow shading highlights an Esg-bound region (EBR), which includes a consensus Esg E-box ([G/A]CAGGTG; Fuse et al, 1994). B, C Amun overexpression in ISC/EBs resembles a reduction in N signalling. (B) Midguts of the indicated genotype were stained with DAPI (nuclei), GFP (ISC/EB) and b-GAL (N activation). (C) CellProfiler quantification of the relative proportion of EE cells in midguts from (B). A similar result was obtained using the milder 5961GS driver (Supplementary Fig S4). ***P < 0.001, Mann–Whitney U-test. Scale bars = 20 lm. D Amun mRNA upregulation following Esg knockdown in ISC/EBs. qPCR measurements of relative transcript abundances for the indicated genes from esg-GFP+ and esg-GFP� cells isolated by FACS sorting from esg-GFP, 5961GS > UAS-esgRNAi flies incubated on ethanol or RU486 (25 lg/ml for 3 days) to induce RNAi expression. Shown are means (� SEM) of efficiency-corrected relative quantities for each primer set, normalized to the corresponding GFP+/EtOH sample and to RpL32 levels (used as reference). * denotes a significant reduction in esg and an increase in Amun transcript levels, …