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• W2191905568 abstract "•Sox21a is specifically required for adult intestinal stem cell proliferation•Sox21a expression is induced in response to tissue damage•Sox21a is controlled by the JNK- and ERK-signaling pathways Adult organs and their resident stem cells are constantly facing the challenge of adapting cell proliferation to tissue demand, particularly in response to environmental stresses. Whereas most stress-signaling pathways are conserved between progenitors and differentiated cells, stem cells have the specific ability to respond by increasing their proliferative rate, using largely unknown mechanisms. Here, we show that a member of the Sox family of transcription factors in Drosophila, Sox21a, is expressed in intestinal stem cells (ISCs) in the adult gut. Sox21a is essential for the proliferation of these cells during both normal epithelium turnover and repair. Its expression is induced in response to tissue damage, downstream of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, to promote ISC proliferation. Although short-lived, Sox21a mutant flies show no developmental defects, supporting the notion that this factor is a specific regulator of adult stem cell proliferation. Adult organs and their resident stem cells are constantly facing the challenge of adapting cell proliferation to tissue demand, particularly in response to environmental stresses. Whereas most stress-signaling pathways are conserved between progenitors and differentiated cells, stem cells have the specific ability to respond by increasing their proliferative rate, using largely unknown mechanisms. Here, we show that a member of the Sox family of transcription factors in Drosophila, Sox21a, is expressed in intestinal stem cells (ISCs) in the adult gut. Sox21a is essential for the proliferation of these cells during both normal epithelium turnover and repair. Its expression is induced in response to tissue damage, downstream of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, to promote ISC proliferation. Although short-lived, Sox21a mutant flies show no developmental defects, supporting the notion that this factor is a specific regulator of adult stem cell proliferation. Resident stem cell populations are essential for the long-term homeostasis of many tissues in organisms ranging from invertebrates to humans. One essential property of these cells is their ability to specifically respond to tissue damage, transiently increasing their proliferation rate to produce new differentiated cells and help restore tissue integrity. Interestingly, the activity of many of the signaling pathways that control this proliferative response in stem cells leads to distinct biological outcomes in non-stem populations. Yet the mechanisms controlling this specificity remain largely unknown in most stem and progenitor populations. Members of the SRY-box (Sox) transcription factor family are defined by the presence of a specific high-mobility-group box domain first identified in the SRY gene. Sox proteins are expressed in many developing tissues and are critical regulators of cell proliferation, differentiation, or establishment of stem and progenitor populations. More recently, the central role of Sox factors in the control of stem cell identity has been highlighted by the identification of Sox2 as one of the factors originally required to reprogram differentiated cells into induced pluripotent stem cells. Aside from their roles in embryonic stem cells, cell reprogramming, and development, expression of Sox transcription factors has been found in many stem or progenitor cell populations in adult tissues, in which it is essential for the maintenance of tissue-specific stem cells and proper differentiation of progenitors (Sarkar and Hochedlinger, 2013Sarkar A. Hochedlinger K. The sox family of transcription factors: versatile regulators of stem and progenitor cell fate.Cell Stem Cell. 2013; 12: 15-30Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). However, in most cases, the mechanisms regulating the function of Sox transcription factors in adult tissues remain largely unknown. In recent years, the adult Drosophila intestine has emerged as a powerful model to study somatic stem cell regulation in vivo (Micchelli and Perrimon, 2006Micchelli C.A. Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium.Nature. 2006; 439: 475-479Crossref PubMed Scopus (821) Google Scholar, Ohlstein and Spradling, 2006Ohlstein B. Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells.Nature. 2006; 439: 470-474Crossref PubMed Scopus (790) Google Scholar). Intestinal stem cells (ISCs) are the only proliferating cells in the fly gut and are essential for the maintenance of the midgut epithelium integrity, metabolic homeostasis, and longevity (Biteau et al., 2011Biteau B. Hochmuth C.E. Jasper H. Maintaining tissue homeostasis: dynamic control of somatic stem cell activity.Cell Stem Cell. 2011; 9: 402-411Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Jiang and Edgar, 2012Jiang H. Edgar B.A. Intestinal stem cell function in Drosophila and mice.Curr. Opin. Genet. Dev. 2012; 22: 354-360Crossref PubMed Scopus (116) Google Scholar). ISC proliferation is tightly controlled by the activity of many signaling pathways during both normal tissue turnover (e.g., insulin and epidermal growth factor/mitogen-activated protein kinase [EGF/MAPK] pathways; Biteau and Jasper, 2011Biteau B. Jasper H. EGF signaling regulates the proliferation of intestinal stem cells in Drosophila.Development. 2011; 138: 1045-1055Crossref PubMed Scopus (227) Google Scholar, Biteau et al., 2010Biteau B. Karpac J. Supoyo S. Degennaro M. Lehmann R. Jasper H. Lifespan extension by preserving proliferative homeostasis in Drosophila.PLoS Genet. 2010; 6: e1001159Crossref PubMed Scopus (264) Google Scholar, Buchon et al., 2010Buchon N. Broderick N.A. Kuraishi T. Lemaitre B. Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection.BMC Biol. 2010; 8: 152Crossref PubMed Scopus (261) Google Scholar, Jiang et al., 2011Jiang H. Grenley M.O. Bravo M.J. Blumhagen R.Z. Edgar B.A. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila.Cell Stem Cell. 2011; 8: 84-95Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and tissue repair in response to oxidative stress, tissue damage, or infection (e.g., Jun N-terminal kinase [JNK], JAK/Stat, and Hippo/Yorkie pathways; Beebe et al., 2010Beebe K. Lee W.C. Micchelli C.A. JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage.Dev. Biol. 2010; 338: 28-37Crossref PubMed Scopus (168) Google Scholar, Biteau et al., 2008Biteau B. Hochmuth C.E. Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut.Cell Stem Cell. 2008; 3: 442-455Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Buchon et al., 2009Buchon N. Broderick N.A. Chakrabarti S. Lemaitre B. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.Genes Dev. 2009; 23: 2333-2344Crossref PubMed Scopus (519) Google Scholar, Jiang et al., 2009Jiang H. Patel P.H. Kohlmaier A. Grenley M.O. McEwen D.G. Edgar B.A. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut.Cell. 2009; 137: 1343-1355Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, Lin et al., 2010Lin G. Xu N. Xi R. Paracrine unpaired signaling through the JAK/STAT pathway controls self-renewal and lineage differentiation of Drosophila intestinal stem cells.J. Mol. Cell Biol. 2010; 2: 37-49Crossref PubMed Scopus (109) Google Scholar, Liu et al., 2010Liu W. Singh S.R. Hou S.X. JAK-STAT is restrained by Notch to control cell proliferation of the Drosophila intestinal stem cells.J. Cell. Biochem. 2010; 109: 992-999PubMed Google Scholar, Ren et al., 2010Ren F. Wang B. Yue T. Yun E.Y. Ip Y.T. Jiang J. Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways.Proc. Natl. Acad. Sci. USA. 2010; 107: 21064-21069Crossref PubMed Scopus (238) Google Scholar, Shaw et al., 2010Shaw R.L. Kohlmaier A. Polesello C. Veelken C. Edgar B.A. Tapon N. The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration.Development. 2010; 137: 4147-4158Crossref PubMed Scopus (235) Google Scholar, Staley and Irvine, 2010Staley B.K. Irvine K.D. Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation.Curr. Biol. 2010; 20: 1580-1587Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, Xu et al., 2011Xu N. Wang S.Q. Tan D. Gao Y. Lin G. Xi R. EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells.Dev. Biol. 2011; 354: 31-43Crossref PubMed Scopus (160) Google Scholar). Yet little is known about the transcriptional network that integrates all these signals into a coordinated proliferative response. The Sox protein family is highly conserved from invertebrates to humans. Eight genes encoding putative Sox transcription factors have been identified in the Drosophila genome (Phochanukul and Russell, 2010Phochanukul N. Russell S. No backbone but lots of Sox: Invertebrate Sox genes.Int. J. Biochem. Cell Biol. 2010; 42: 453-464Crossref PubMed Scopus (63) Google Scholar). The function of SoxNeuro and Dichaete in embryonic development and the developing nervous system is best characterized, whereas Sox100B was identified as a critical regulator of male germline specification (Buescher et al., 2002Buescher M. Hing F.S. Chia W. Formation of neuroblasts in the embryonic central nervous system of Drosophila melanogaster is controlled by SoxNeuro.Development. 2002; 129: 4193-4203PubMed Google Scholar, Nanda et al., 2009Nanda S. DeFalco T.J. Loh S.H. Phochanukul N. Camara N. Van Doren M. Russell S. Sox100B, a Drosophila group E Sox-domain gene, is required for somatic testis differentiation.Sex Dev. 2009; 3: 26-37Crossref PubMed Scopus (40) Google Scholar, Soriano and Russell, 1998Soriano N.S. Russell S. The Drosophila SOX-domain protein Dichaete is required for the development of the central nervous system midline.Development. 1998; 125: 3989-3996PubMed Google Scholar). However, the potential function of Sox factors in adult somatic stem cell populations has not yet been investigated. Here, we show that Sox21a, one of the Drosophila Sox2 homologs, is a critical regulator of ISC function in the adult fly. We found that its expression is required for epithelial turnover and is regulated by the JNK and extracellular signal-regulated kinase (ERK) pathways to control ISC proliferation in response to tissue damage. Importantly, Sox21a is dispensable during development, demonstrating that its function represents a novel mechanism regulating cell proliferation specifically in adult stem cells. In order to investigate the potential role of Sox transcription factors in the regulation of ISC function, we first asked whether members of this gene family are expressed in the fly intestinal epithelium. Using in situ hybridization, we found that the Sox21a mRNA is exclusively detected in esg-positive ISCs and enteroblasts (EBs) in the adult fly intestine (Figure 1A). To confirm that the Sox21a protein is expressed in these cells, we developed a polyclonal antibody against this factor. This antibody specifically recognizes Sox21a protein in the nuclei of diploid cells, distinct from the prospero-positive endocrine cells (EEs) and polyploid enterocytes (ECs) (Figure 1B). We confirmed that these cells are ISCs and EBs using specific markers. Both ISCs and EBs express the escargot marker, whereas ISCs express the Notch ligand Delta (Delta-LacZ; Figure 1C) and EBs show high activity for the Notch reporter GBE-Su(H)-LacZ (Figure 1D; Ohlstein and Spradling, 2007Ohlstein B. Spradling A. Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling.Science. 2007; 315: 988-992Crossref PubMed Scopus (487) Google Scholar). As suggested by the result of our in situ hybridization analysis of Sox21a expression, the Sox21a protein is detected in both ISCs and EBs throughout the entire midgut epithelium (Figures 1C and 1D). To further confirm the specificity of the observed signal, we expressed a dsRNA directed against Sox21a using the temperature-sensitive esgGal4ts driver. This knockdown is sufficient to abolish Sox21a expression in the intestine, as shown by western blot using total protein extracts from dissected guts (Figure 1E), confirming that Sox21a expression is restricted to esg-positive cells in the Drosophila intestine. Altogether, these results demonstrate that Sox21a is specifically expressed in ISCs and EBs in the intestinal epithelium. The Sox21a expression pattern in the adult gut strongly suggests that this transcription factor specifically functions in ISCs. Therefore, we tested whether Sox21a is required for stem cell proliferation. To this end, we identified a transposable element insertion in the Sox21a locus (Sox21af04672) that strongly impairs Sox21a expression in the intestine (Figure 2A), without affecting the proportion of ISCs or EEs in the gut epithelium (Figure S1A). We assessed the effect of this mutation on ISC proliferation after tissue damage. Exposure to dextran sulfate sodium (DSS) induces a robust proliferative response that can be easily quantified by counting the number of cells positive for the mitotic marker phospho-histone H3 (pH3) in the midgut (Amcheslavsky et al., 2009Amcheslavsky A. Jiang J. Ip Y.T. Tissue damage-induced intestinal stem cell division in Drosophila.Cell Stem Cell. 2009; 4: 49-61Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Consistent with previous reports, DSS induces ISC proliferation in wild-type or heterozygous animals; however, this response is abolished in Sox21af04672 homozygous flies (Figure 2B), suggesting that Sox21a is essential for ISC proliferation. We have previously shown that flies with greatly impaired ISC proliferative capacity are short lived (Biteau et al., 2010Biteau B. Karpac J. Supoyo S. Degennaro M. Lehmann R. Jasper H. Lifespan extension by preserving proliferative homeostasis in Drosophila.PLoS Genet. 2010; 6: e1001159Crossref PubMed Scopus (264) Google Scholar). Thus, to test the functional requirement for Sox21a, we backcrossed the Sox21af04672 allele in two different genetic backgrounds and analyzed the lifespan of control, heterozygous, and homozygous mutant animals. Consistent with the critical role of Sox21a in intestinal homeostasis, we found that Sox21af04672 homozygous females are significantly shorter lived than their siblings (Figures S1B and S1C). To better characterize this Sox21a loss-of-function proliferation defect, we next analyzed ISC lineages by generating positively marked stem cell clones (MARCM; Lee and Luo, 1999Lee T. Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.Neuron. 1999; 22: 451-461Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar) in the adult posterior midgut. Consistent with previous studies, control clones reach an average size of 8 to 12 cells/clones, 7 days after induction (Figure 2C). However, clones expressing two distinct RNAi constructs directed against Sox21a are much smaller than their respective controls (Figure 2C), demonstrating that ISCs in which Sox21a is knocked down are essentially incapable of proliferation. Of note, comparable numbers of clones were observed in all conditions (data not shown) and Sox21aRNAi-expressing single-cell clones retain Delta expression (Figure 2C), confirming that Sox21a knockdown specifically impairs ISCs proliferation but does not affect their survival or self-renewal. To support these results, we used the esgGal4ts driver to specifically express three independent RNAi constructs in all ISCs and EBs and found that these manipulations are sufficient to reduce ISC proliferation under normal conditions and strongly inhibit the DSS-induced proliferative response (Figure 2D). We show that Sox21a is expressed in both ISCs and EBs (Figures 1C and 1D). Previous studies have reported that defects in EBs are capable of signaling back to the ISCs, preventing further stem cell division (Choi et al., 2011Choi N.H. Lucchetta E. Ohlstein B. Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway.Proc. Natl. Acad. Sci. USA. 2011; 108: 18702-18707Crossref PubMed Scopus (109) Google Scholar). To exclude such non-cell-autonomous effect, we tested whether Sox21a is required in ISCs themselves to permit cell proliferation. We expressed the Sox21aRNAi(KK) construct using the ISC-specific Delta-Gal4ts driver and the EB-specific GBE-Su(H)-Gal4ts driver (Zeng et al., 2010Zeng X. Chauhan C. Hou S.X. Characterization of midgut stem cell- and enteroblast-specific Gal4 lines in Drosophila.Genesis. 2010; 48: 607-611Crossref PubMed Scopus (110) Google Scholar). Similar to what we observed using the esgGal4ts driver, knockdown of Sox21a in ISCs only is sufficient to significantly impair cell proliferation in response to DSS, whereas Sox21a knockdown in EBs does not affect ISCs proliferation in these conditions (Figure 2E). Altogether, these results demonstrate that Sox21a is specifically required in ISCs to maintain their proliferative capacity under homeostatic conditions and during tissue repair. The essential role of Sox21a in ISC proliferation prompted us to investigate the possibility that its expression is regulated to control the stem cell stress response. We found that Sox21a protein level in the intestine increases dramatically after DSS treatment (Figures 3A, S2A, and S2B). Importantly, this stress-induced expression is absent in esgGal4 > Sox21aRNAi animals, confirming that, even under stress conditions, Sox21a expression is limited to ISCs and EBs. Previous studies by us and others have shown that the population of esg-positive cells expands after exposure to stress or in aging flies (Amcheslavsky et al., 2009Amcheslavsky A. Jiang J. Ip Y.T. Tissue damage-induced intestinal stem cell division in Drosophila.Cell Stem Cell. 2009; 4: 49-61Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Biteau et al., 2008Biteau B. Hochmuth C.E. Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut.Cell Stem Cell. 2008; 3: 442-455Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Buchon et al., 2009Buchon N. Broderick N.A. Chakrabarti S. Lemaitre B. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.Genes Dev. 2009; 23: 2333-2344Crossref PubMed Scopus (519) Google Scholar, Jiang et al., 2009Jiang H. Patel P.H. Kohlmaier A. Grenley M.O. McEwen D.G. Edgar B.A. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut.Cell. 2009; 137: 1343-1355Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). To confirm that the observed Sox21a induction is caused by an increased expression in ISCs, rather than an increased number of esg-positive Sox21a-expressing cells, we exposed flies to DSS for short periods of time and detected increased Sox21a protein level as early as 24 hr, a time when no supernumerary esg-positive cells are present in the intestinal epithelium, as confirmed by similar GFP expression levels (Figure 3B). Similar induction was observed when flies are exposed to paraquat, a compound that leads to the production of reactive oxygen species in the intestinal epithelium and increases ISC proliferation (Biteau et al., 2008Biteau B. Hochmuth C.E. Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut.Cell Stem Cell. 2008; 3: 442-455Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Choi et al., 2008Choi N.H. Kim J.G. Yang D.J. Kim Y.S. Yoo M.A. Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor.Aging Cell. 2008; 7: 318-334Crossref PubMed Scopus (189) Google Scholar; Figure 3C). In parallel to our western blot analysis, we used a blind scoring approach to evaluate the intensity of the Sox21a immuno-staining in the intestine of control and DSS- or paraquat-treated flies. We found a greater proportion of intestines showing moderate to high Sox21a protein level in stressed animals compared to untreated animals (Figures 3D and S2C). In addition, we used immuno-staining to show that the expression of Sox21a protein is uniquely induced in esg-positive cells after DSS exposure and used the ISC-specific marker Delta to confirm that Sox21a expression specifically increases in ISCs and EBs in response to tissue damage (Figures 3E and S2D). Finally, we show that Sox21a mRNA level is induced in response to DSS, suggesting that this factor is regulated at the transcriptional level (Figure 3F). This regulation strongly suggested that elevated Sox21a protein level promotes ISC proliferation during tissue repair. To test this notion, we first overexpressed the endogenous Sox21a gene using an EP line (Sox21ad03399; a P-element carrying UAS sites inserted in the Sox21a promoter region). When combined with the esgGal4ts driver, this insertion is sufficient to significantly induce Sox21a mRNA level (Figure 3G) and leads to a robust increase in cell proliferation in the intestine (Figure 3H). In addition, we generated a UAS-driven Flag-tagged Sox21a transgene. Expression of this fusion protein was confirmed by western blot and immunochemistry (Figure 3I) and is sufficient to promote ISCs proliferation when driven by the esgGal4 (ISC/EBs) or the DeltaGal4 (ISCs only) driver (Figure 3J). Finally, although Sox21a-overexpressing MARCM clones grow larger than control (Figure S3A), we found no evidence that Sox21a overexpression affects the ability of the ISC lineage to differentiate into EEs and ECs (Figures S3B–S3D). Collectively, these results demonstrate that Sox21a expression is induced in ISCs to promote cell proliferation in response to tissue damage. Previous studies have established that ISC proliferation requires the activity of multiple signaling pathways (Biteau et al., 2011Biteau B. Hochmuth C.E. Jasper H. Maintaining tissue homeostasis: dynamic control of somatic stem cell activity.Cell Stem Cell. 2011; 9: 402-411Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Jiang and Edgar, 2012Jiang H. Edgar B.A. Intestinal stem cell function in Drosophila and mice.Curr. Opin. Genet. Dev. 2012; 22: 354-360Crossref PubMed Scopus (116) Google Scholar, Pasco et al., 2015Pasco M.Y. Loudhaief R. Gallet A. The cellular homeostasis of the gut: what the Drosophila model points out.Histol. Histopathol. 2015; 30: 277-292PubMed Google Scholar). The JNK and EGF/Ras/ERK pathways are two critical components of the regulatory network involved in the maintenance of ISC proliferative capacity during normal tissue turnover and essential for the increased proliferation under stress conditions (Biteau et al., 2008Biteau B. Hochmuth C.E. Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut.Cell Stem Cell. 2008; 3: 442-455Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, Biteau and Jasper, 2011Biteau B. Jasper H. EGF signaling regulates the proliferation of intestinal stem cells in Drosophila.Development. 2011; 138: 1045-1055Crossref PubMed Scopus (227) Google Scholar, Buchon et al., 2010Buchon N. Broderick N.A. Kuraishi T. Lemaitre B. Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection.BMC Biol. 2010; 8: 152Crossref PubMed Scopus (261) Google Scholar, Jiang et al., 2011Jiang H. Grenley M.O. Bravo M.J. Blumhagen R.Z. Edgar B.A. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila.Cell Stem Cell. 2011; 8: 84-95Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, Xu et al., 2011Xu N. Wang S.Q. Tan D. Gao Y. Lin G. Xi R. EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells.Dev. Biol. 2011; 354: 31-43Crossref PubMed Scopus (160) Google Scholar). The expression of Sox21a and its function in the control of ISC proliferation lead us to investigate the potential role of Sox21a downstream of JNK and Ras signaling. To this end, we first genetically induced ISCs proliferation by activating the JNK and Ras/ERK pathways through overexpression of JNKK/Hep or expression of activated Ras (RasV12) under the control of the esgGal4ts driver. In both conditions, the expression of the Sox21a protein in the gut is dramatically increased (Figures 4A and S4A), recapitulating the induction observed in response to stress. Next, because JNK and Ras are critical for stress-mediated proliferation (Figure 4B), we asked whether these pathways are required for Sox21a expression in response to DSS. We found that expressing a dominant-negative form of JNK/Bsk or an RNAi directed against Ras using the esgGal4ts driver is sufficient to abolish Sox21a expression in the intestine of DSS-treated animals (Figure 4C). Last, we tested whether Sox21a is essential for Hep- and Ras-induced proliferation and found that knocking down Sox21a prevents the hyperproliferation induced by overexpression of Hep/JNKK or RasV12 (Figures 4D and S4B), confirming that Sox21a is a critical mediator of JNK and Ras signaling in the control of ISC proliferation. We have previously established that the AP-1 transcription factor Fos (kayak in Drosophila) integrates the activity of both JNK and Ras/ERK signaling in ISCs and is essential for proliferation downstream of these pathways (Biteau and Jasper, 2011Biteau B. Jasper H. EGF signaling regulates the proliferation of intestinal stem cells in Drosophila.Development. 2011; 138: 1045-1055Crossref PubMed Scopus (227) Google Scholar; Figure S4B). Thus, we hypothesized that Sox21a is an essential target of Fos for the control of ISC proliferative rate. To test this notion, we first exposed flies that expressed RNAi constructs directed against Fos in esg-positive cells to DSS. In the intestine of these animals, we found that the DSS-mediated induction of Sox21a protein is strongly inhibited and that this inhibition correlates with the efficacy of the Fos knockdown (Figure 4E). Consistent with this result, we also found that Fos is essential for Hep- and Ras-mediated Sox21a expression (Figure 4F). Finally, we reasoned that, if Sox21a is a major target of the Ras/ERK pathway and Fos in the regulation of ISC proliferation, ectopic expression of Sox21a might be sufficient to bypass the requirement for these signaling components. To test this hypothesis, we simultaneously expressed the Sox21aFlag construct with either RasRNAi or FosRNAi in esg-positive cells. Whereas RasRNAi fully inhibits DSS-mediated proliferation (Figure 4B), we found that Sox21a expression partially but significantly rescues this proliferation defect (Figure 4G). Similarly, although FosRNAi completely blocks Hep- and Ras- mediated proliferation (Figure S4B), Fos knockdown has little to no effect on Sox21a-mediated proliferation (Figure 4H). Together, these results support a model in which Sox21a expression is controlled by Ras, JNK, and Fos to promote ISC proliferation (Figure 4I). In this work, we demonstrate that Sox21a, a member of the Sox2 sub-family of transcription factors, is essential for cell proliferation in the adult Drosophila intestine under homeostatic conditions and in response to stress. Strikingly, although we found that Sox21a mutant adult flies have dramatic ISCs proliferation defects and are short lived (Figure S1), they do not display any visible developmental phenotype, recapitulating a reported analysis of null Sox21a mutants (Phochanukul and Russell, 2010Phochanukul N. Russell S. No backbone but lots of Sox: Invertebrate Sox genes.Int. J. Biochem. Cell Biol. 2010; 42: 453-464Crossref PubMed Scopus (63) Google Scholar). Thus, this demonstrates that ISCs use stem-cell-specific mechanisms to control cell proliferation. Further studies will be required to understand how Sox21a interacts with other transcription factors that have been shown to regulate ISC proliferation, such as Myc, Nrf2, Stat92E, and Yorkie (Amcheslavsky et al., 2011Amcheslavsky A. Ito N. Jiang J. Ip Y.T. Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells.J. Cell Biol. 2011; 193: 695-710Crossref PubMed Scopus (76) Google Scholar, Beebe et al., 2010Beebe K. Lee W.C. Micchelli C.A. JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage.Dev. Biol. 2010; 338: 28-37Crossref PubMed Scopus (168) Google Scholar, Buchon et al., 2009Buchon N. Broderick N.A. Chakrabarti S. Lemaitre B. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.Genes Dev. 2009; 23: 2333-2344Crossref PubMed Scopus (519) Google Scholar, Hochmuth et al., 2011Hochmuth C.E. Biteau B. Bohmann D. Jasper H. Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila.Cell Stem Cell. 2011; 8: 188-199Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, Jiang et al., 2009Jiang H. Patel P.H. Kohlmaier A. Grenley M.O. McEwen D.G. Edgar B.A. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut.Cell. 2009; 137: 1343-1355Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, Lin et al., 2010Lin G. Xu N. Xi R. Paracrine unpaired signaling through the JAK/STAT pathway controls self-renewal and lineage differentiation of Drosophila intestinal stem cells.J. Mol. Cell Biol. 2010; 2: 37-49Crossref PubMed Scopus (109) Google Scholar, Liu et al., 2010Liu W. Singh S.R. Hou S.X. JAK-STAT is restrained by Notch to control cell proliferation of the Drosophila intestinal stem cells.J. Cell. Biochem. 2010; 109: 992-999PubMed Google Scholar, Ren et al., 2010Ren F. Wang B. Yue T. Yun E.Y. Ip Y.T. Jiang J. Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways.Proc. Natl. Acad. Sci. USA. 2010; 107: 21064-21069Crossref PubMed Scopus (238) Google Scholar, Shaw et al., 2010Shaw R.L. Kohlmaier A. Polesello C. Veelken C. Edgar B.A. Tapon N. The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration.Development. 2010; 137: 4147-4158Crossref PubMed Scopus (235) Google Scholar, Staley and Irvine, 2010Staley B.K. Irvine K.D. Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation.Curr. Biol. 2010; 20: 1580-1587Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In addition, we anticipate that the identification of Sox21a transcriptional targets in ISCs will be required to fully decipher the mechanism by which this factor controls ISC cell cycle and/or quiescent state. This also constitutes a unique opportunity to study the adult-specific functions of a Sox factor, apart from their requirement during development. Here, we show that JNK and Ras/ERK signaling, as well as the AP-1 transcription factor Fos, are required for Sox21a induction in response to tissue damage. Although our data support a model in which the activity of Fos is regulated by JNK and ERK to control Sox21a expression (Figure 4I), further studies will be necessary to investigate the potential mechanisms of such regulation and test whether Fos directly binds to the Sox21a locus and controls its transcription. Whereas the transcriptional response to various stresses or the activation of these pathways has been investigated in developing tissues and other adult organs, Sox21a has not been identified as a target of these pathways (Asha et al., 2003Asha H. Nagy I. Kovacs G. Stetson D. Ando I. Dearolf C.R. Analysis of Ras-induced overproliferation in Drosophila hemocytes.Genetics. 2003; 163: 203-215Crossref PubMed Google Scholar, Girardot et al., 2004Girardot F. Monnier V. Tricoire H. Genome wide analysis of common and specific stress responses in adult Drosophila melanogaster.BMC Genomics. 2004; 5: 74Crossref PubMed Scopus (155) Google Scholar, Wang et al., 2003Wang M.C. Bohmann D. Jasper H. JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila.Dev. Cell. 2003; 5: 811-816Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Thus, our findings suggest that unidentified stem-cell-specific factor(s) cooperate with Fos to control Sox21a expression in ISCs and EBs. Additional work will be necessary to carefully describe the regulation of Sox21a and the possible role of ISC-specific factors, such as esg (Korzelius et al., 2014Korzelius J. Naumann S.K. Loza-Coll M.A. Chan J.S. Dutta D. Oberheim J. Gläßer C. Southall T.D. Brand A.H. Jones D.L. Edgar B.A. Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells.EMBO J. 2014; 33: 2967-2982Crossref PubMed Scopus (77) Google Scholar, Micchelli and Perrimon, 2006Micchelli C.A. Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium.Nature. 2006; 439: 475-479Crossref PubMed Scopus (821) Google Scholar). It will also be interesting to test whether other signaling pathways, such as JAK/STAT and Hippo/Yorkie, are involved in the regulation of Sox21a expression. The identification of potential common transcriptional targets will help to understand how these signaling pathways crosstalk in ISCs and how different signals are integrated into a coordinated proliferative response. Interestingly, like the activation of stress-signaling pathways, expression of Sox factors is essential for tumor formation in many tissues (Gracz and Magness, 2011Gracz A.D. Magness S.T. Sry-box (Sox) transcription factors in gastrointestinal physiology and disease.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G503-G515Crossref PubMed Scopus (22) Google Scholar, Liu et al., 2013Liu K. Lin B. Zhao M. Yang X. Chen M. Gao A. Liu F. Que J. Lan X. The multiple roles for Sox2 in stem cell maintenance and tumorigenesis.Cell. Signal. 2013; 25: 1264-1271Crossref PubMed Scopus (193) Google Scholar). Therefore, it will be interesting to test whether, similarly to the regulation we describe here in ISCs, stress pathways, such as JNK and Ras/ERK, directly control the expression of Sox factor(s) in mammals. In this context, our results may provide new insights in the mechanisms that control tissue repair and tumorigenesis in higher organisms, including in humans. Additional experimental procedures are described in Supplemental Information. The esgGal4, DeltaGal4, and Su(H)GBEGal4 drivers were combined with a ubiquitously expressed temperature-sensitive Gal80 inhibitor (tubGal80ts). Crosses and flies were kept at 18°C (permissive temperature) and 3- to 5-day-old females were then shifted to 29°C for 2 or 3 days to allow expression of the transgenes before analysis or additional treatment. In order to induce UAS-driven gene expression with the Act5CGeneswitch driver, food vials were supplemented with 100 μl of a 5 mg/ml solution of mefiprestone, resulting in a final concentration of 0.2 mg/ml. Positively marked clones were generated by somatic recombination using the following MARCM stocks: hsFlp;FRT40A,tubGal80;tubGal4,UAS-GFP (MARCM40A; gift from B. Ohlstein) and hsFlp,UAS-GFP;;tubGal4,FRT82B,tubGal80 (MARCM82B). Three- to five-day-old mated female flies were heat shocked for 45 min at 37°C to induce somatic recombination. Flies were transferred to 25°C, and clones were observed 7 days after induction. Only isolated ISC clones in the posterior midgut were included in our analysis. For all stress experiments, young mated females were cultured on standard food. Flies were starved for 6 hr in empty vials and re-fed with a 5% sucrose (AMRESCO) solution with or without 5 mM paraquat (Sigma-Aldrich) or 4% DSS (Sigma-Aldrich; 9 KDa∼20 KDa). Flies were then dissected at the indicated time points for western analysis and immunocytochemistry. Intact guts were dissected in PBS and proteins extracted in Laemmli buffer, separated on 10% acrylamide gel and transferred according to standard procedures. Antibodies directed against β-actin (Cell Signaling Technology; 1:5,000 dilution), Flag tag (Sigma; 1:5,000 dilution), and GFP (Invitrogen; 1:5,000 dilution) were used and Sox21a (this study; 1:50,000 dilution). Total proteins were extracted from 12 guts, and the equivalent of 1.2, 4.8, and 4.8 guts was used for β-actin, Sox21a, and GFP detection, respectively. Total RNA from eight dissected guts from young mated females or three whole flies was extracted using Trizol (Invitrogen), according to manufacturer instructions. cDNA was synthesized using an oligo-dT primer. Real-time PCR was performed on a Bio-Rad iQ5 detection system using the following primers: Sox21a forward 5′-GCCGAGTGGAAATTACTCACCGAA-3′; Sox21a reverse 5′-TGCGACGTGGTCGATACTTGTAGT-3′; actin5c forward 5′-CTCGCCACTTGCGTTTACAGT-3′; and actin5c reverse 5′-TCCATATCGTCCCAGTTGGTC-3′. Relative expression was calculated using the ΔΔCt method and normalized to actin5c levels. In situ hybridization protocol was adapted from previously described procedure (Lécuyer et al., 2008Lécuyer E. Parthasarathy N. Krause H.M. Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.Methods Mol. Biol. 2008; 420: 289-302Crossref PubMed Scopus (94) Google Scholar). An ∼550-bp sequence of the Sox21a cDNA was amplified, using the 5′-GCCGAGTGGAAATTACTCACCGAA-3′ and 5′-AGGGTGGAGTTTCCGGACTTATCA-3′ primers, and cloned in the pCRII-TOPO vector to generate the antisense RNA probe. Intact fly intestines were dissected in PBS and fixed at room temperature for 45 min in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde. Tissues were blocked in PBS, 0.5% BSA, and 0.1% Triton X-100 and incubated in the same buffer at 4°C. For Delta and Sox21a staining, dissected intestines were fixed in PBS+4% formaldehyde, dehydrated with 100% methanol, and progressively rehydrated in the staining buffer. The anti-Delta (1:100 dilution) and anti-Prospero (1:250 dilution) were obtained from the Developmental Studies Hybridoma Bank and the anti-phosphoHistoneH3 (1:2,000 dilution) from Millipore. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. Hoechst was used to stain DNA. Confocal images were collected using a Leica SP5 confocal system and processed using the Leica software and Adobe Photoshop CS5. For all experiments, the data are represented as average ± SEM. All p values are calculated using unpaired two-tailed Student’s t test unless stated otherwise. This work was initiated in the laboratory of Prof. Heinrich Jasper at the University of Rochester, and we sincerely thank him for his continuous support. We are grateful to Dirk Bohmann for his comments on our manuscript and M. Nuzzo for technical assistance and helping with the generation of the anti-Sox21a antibody. This work was funded in part by a New Scholar in Aging Award from the Ellison Medical Foundation to B.B. (AG-NS-0990-13). Download .pdf (.8 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figures S1–S4" @default.
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• W2191905568 title "A Sox Transcription Factor Is a Critical Regulator of Adult Stem Cell Proliferation in the Drosophila Intestine" @default.
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