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• W2021181797 abstract "Article30 April 2014Open Access c-Src drives intestinal regeneration and transformation Julia B Cordero Julia B Cordero The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Rachel A Ridgway Rachel A Ridgway The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Nicola Valeri Nicola Valeri Institute of Cancer Research, Sutton, London, UK Search for more papers by this author Colin Nixon Colin Nixon The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Margaret C Frame Margaret C Frame Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author William J Muller William J Muller Goodman Cancer Research Center, McGill University, Montreal, QC, Canada Search for more papers by this author Marcos Vidal Marcos Vidal The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Owen J Sansom Corresponding Author Owen J Sansom The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Julia B Cordero Julia B Cordero The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Rachel A Ridgway Rachel A Ridgway The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Nicola Valeri Nicola Valeri Institute of Cancer Research, Sutton, London, UK Search for more papers by this author Colin Nixon Colin Nixon The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Margaret C Frame Margaret C Frame Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author William J Muller William J Muller Goodman Cancer Research Center, McGill University, Montreal, QC, Canada Search for more papers by this author Marcos Vidal Marcos Vidal The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Owen J Sansom Corresponding Author Owen J Sansom The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK Search for more papers by this author Author Information Julia B Cordero1,5, Rachel A Ridgway1, Nicola Valeri2, Colin Nixon1, Margaret C Frame4, William J Muller3, Marcos Vidal1 and Owen J Sansom 1 1The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK 2Institute of Cancer Research, Sutton, London, UK 3Goodman Cancer Research Center, McGill University, Montreal, QC, Canada 4Edinburgh Cancer Research Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK 5Present address: Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden, Glasgow, UK *Corresponding author: Tel: +44 141 330 3953; E-mail: [email protected] The EMBO Journal (2014)33:1474-1491https://doi.org/10.1002/embj.201387454 See also: P Sousa-Victor & H Jasper et al (July 2014) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo. Synopsis These comprehensive, cross-organismal experiments determine the evolutionary conservation and central position of Src family kinases in intestinal stem cell renewal, tumour formation and proliferative tissue regeneration. Src is activated in stem/progenitor cells in response to damage and loss of Apc in the intestine. Src is required for intestinal regeneration and Apc loss-driven tumourigenesis. Src family kinases are required for homeostatic self-renewal of the intestinal epithelium. Src requirement in intestine physiology and pathology is conserved from Drosophila to mammals. Introduction Since its discovery in the 1970s, the non-receptor tyrosine kinase Src has been implicated in multiple types of human cancers (Irby & Yeatman, 2000). There is rationale for a driver role for Src within CRC, and putative Src activating mutations were reported specifically within CRC, with amplification found in up to 20% of the advanced human CRC tumours (Irby et al, 1999; The Cancer Genome Atlas Network, 2012; www.cbioportal.org/public-portal). There is also increased Src activity in progressive stages of CRC (Talamonti et al, 1993; Termuhlen et al, 1993; Jones et al, 2002). Consistent with this, multiple studies have shown high percentage Src activation in CRC and suggested that this upregulation and/or hyperactivation of Src contributes to CRC progression and metastasis (Yeatman, 2004). Studies using Src family kinases (SFKs) inhibitors within CRC cell lines have predominantly shown an impact on invasion rather than proliferation (Serrels et al, 2006). One of the difficulties in interpreting the results with SFK inhibitors is the broad number of targets they inhibit. Given the strong evidence for a role for Src in CRC, it is somewhat surprising that Src inhibitors do not inhibit tumour cell proliferation. Therefore, we here performed genetic studies to more definitely address Src's role in the intestine. CRC is one of the most common cancers and the third most common cause of cancer deaths in the western world. Inactivating mutations in the gene encoding for the negative regulator of Wnt signalling, adenomatous polyposis coli (Apc), are detected in 80% of hereditary and sporadic forms of CRC (Kinzler et al, 1991; Korinek et al, 1997). Mouse models have shown that inactivation of Apc is sufficient to drive intestinal hyperplasia (Sansom et al, 2004; Andreu et al, 2005). Moreover, Apc deletion within the murine intestinal stem cells (ISCs) results in rapid adenoma formation suggesting they can act as cells of origin in CRC (Barker et al, 2009). Wnt signalling is also required during intestinal regeneration, and this process mimics many of the features of Apc loss, further suggesting that stem cell proliferation, for example during regeneration, and cancer initiation are linked and co-regulated by key signalling molecules (Cordero & Sansom, 2012; Cordero et al, 2012a,b). The adult Drosophila midgut resembles the vertebrate intestine (Casali & Batlle, 2009), and it has proven to be an extremely useful model to study intestinal homeostasis, regeneration and disease (Biteau et al, 2011; Jiang & Edgar, 2012; Seton-Rogers, 2013). The fly intestinal epithelium is self-renewed by dedicated ISCs (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006). Upon division, ISCs give rise to an undifferentiated progenitor, the enteroblast (EB), which differentiate into either the secretory cell linage, enteroendocrine cells (ee) or the absorptive epithelial cell linage represented by the enterocytes (EC). Importantly, recent work from ourselves and others demonstrated that loss of Apc from the fly midgut results in ISC hyperproliferation (Lee et al, 2009; Cordero et al, 2012a) and recapitulates several hallmarks from mouse models and human CRC (Sansom et al, 2004, 2007; Cordero et al, 2012a). Unlike the nine SFKs encoded in mammalian genomes, there are only two Src family kinase members in Drosophila, Src42A and Src64B as well as a single fly orthologue of the mammalian Src inhibitor, COOH-terminal Src kinase (Csk) and its paralogue, the C-terminal Src kinase homologous kinase (Chk). Hyperactivation of Src in developing Drosophila tissues leads to a wide range of outcomes including cell migration, perturbed differentiation, hyperproliferation and apoptosis (Vidal et al, 2006, 2007). Indeed, the precise level of Src deregulation is very important to the phenotypic outcome, as the relative levels of Src overexpression compared to surrounding normal cells are vital for Drosophila development and mammalian cells in culture (Vidal et al, 2007; Kajita et al, 2010). However, the mechanism of action of Src in Drosophila and mammals in vivo is still poorly understood, particularly how it contributes to intestinal homeostasis. Here, we use Drosophila and mouse genetic models to directly address the role of Src within the intestinal epithelium. We show that Src is necessary and sufficient to drive ISC proliferation during normal tissue homeostasis, damage-induced regeneration and tumour development in vivo. We define, for the first time, key roles for EGFR/Ras/MAPK and Stat signalling as functional mediators of Src-dependent ISC proliferation. Results Ectopic Src activation is observed in mouse models of CRC Src is either hyperactivated or amplified in human CRCs (Yeatman, 2004). We tested whether this was also the case in the mouse intestine. We stained tissue sections from mouse small intestines with an antibody to detect the activated form of Src (pSrc), which cross-reacts with other p-SFKs (Fig 1A–D). Intestines from control animals showed membrane pSrc staining, which was largely restricted to the proliferative ‘crypt’ region of the intestinal epithelium (Fig 1A). No pSrc staining was observed in tissues form mice bearing conditional depletion of Src from the intestinal epithelium combined with constitutive loss of related kinases Fyn and Yes (AhCre Srcfl/fl; Fyn−/−; Yes−/−; Fig 1B). These results confirm the specificity of the antibody to pSFKs in the mouse intestine and indicate that Src, Fyn and Yes are the major SFKs expressed in this tissue. We next analysed the pSrc levels in mouse models of CRC. While we observed no change in Src transcription (data not shown), pSrc immunoreactivity was significantly expanded throughout the hyperproliferative ‘crypt progenitor cell-like’ domain of the intestinal epithelium from mice subject to acute loss of Apc (Fig 1C, dotted line; Sansom et al, 2004). Similarly, ectopic pSrc staining was observed within the core of intestinal polyps from ApcMin/+ mice (100%; n = 10; Fig 1D). To visualize Src activation at the early stages of human CRC, we stained non-invasive human adenomas for pSrc and found it consistently upregulated (100%; n = 7; Fig 1E and F). Therefore, ectopic activation of SFK is detected from the earliest stages of transformation following Apc loss, prior to the formation of invasive tumours and dissemination. This suggested that SFKs might have important roles in the initial stages of tumourigenesis in addition to their recognized roles in invasion and metastasis (Yeatman, 2004). Figure 1. Src upregulation drives ISC proliferation A–D. Immunohistochemistry to detect the activated form of Src (pSrc) in tissue sections from mouse and human intestines. pSrc is detected in the proliferative ‘crypt’ region (indicated with dashed line) at the base of the mouse small intestinal epithelium in control animals (A). Conditional knockout of Src (Srcfl/fl) combined with full knockout of related kinases Fyn and Yes, resulted in no staining within the intestinal epithelium (B). Intestines with conditional Apc knockout (Apcfl/fl) depict the expected ‘crypt-like’ progenitor phenotype (dashed line) and expansion of the pSrc domain (C). Example of a small intestinal polyp from an ApcMin/+ mouse, showing high p-Src staining within the core of the polyp (arrow), is shown in (D). Note normal tissue around polyp showing pSrc localized at the crypt base (dashed line). Scale bars, 100 μm. E, F. pSrc is upregulated within benign human intestinal adenoma lesions (arrows) when compared with normal surrounding tissue (asterisks). Scale bars, 100 μm. G, H. Adult Drosophila midguts overexpressing gfp (G; control) or Src (H) for 7 days under the stem/progenitor cell (ISCs/EBs) driver escargot-gal4 (esgts > gfp and esgts > Src64wt, respectively). Unless otherwise noted green marks esg > gfp cells and Dapi (blue) stains all cell nuclei. Scale bars, 100 μm. I, I'. Paraffin-embedded sections from 7-day-old Src-overexpressing midguts (esgts > Src64wt) analysed by Haematoxylin and Eosin H+E (I) and BrdU (I') staining. Arrows point to ‘polyp-like’ structures containing BrdU+ve cells. J–K'. 7-day-old esgts > Src64wt posterior midguts stained with anti-pH3 (red) to visualize proliferating ISCs (arrows). Scale bars, 20 μm. L. ISC proliferation quantified as the number of cells which stained positive for phosphorylated histone 3 (pH3) in posterior midguts from control animals (esgts > gfp) or animals overexpressing Src (esgts > Scr64wt or esgts > Src42CA) or RNA interference for the Src inhibitor Csk (esgts > Csk-IR). Note that co-expression of human ChK (esgts > Scr64wt; Chk) suppresses Src-driven hyperproliferation in the Drosophila midgut. M–O. 7-day-old adult posterior midguts from animals of the indicated genotypes stained with anti-Delta (red) to label ISCs. Scale bars, 20 μm. P. Quantification of the number of Delta+ve ISCs per field from posterior midguts as in (M-O). Data information: Data in (L) and (P) represent average values ± SEM (***P < 0.0001 one-way ANOVA with Bonferroni's multiple comparison test) Download figure Download PowerPoint Src overexpression is sufficient to drive intestinal hyperproliferation We next asked whether elevated SFK activity was a driver of intestinal hyperproliferation, or a passive event. To address this, we used the adult intestine of Drosophila melanogaster, which was previously validated as a model system to study aspects of CRC (Cordero et al, 2009, 2012a; Lee et al, 2009). Unlike the mouse intestine, which contains proliferating stem- and transit-amplifying (TA) cells, only the stem cells are proliferative in the adult Drosophila midgut (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006). Therefore, assessment of mitotic proliferation in the fly midgut represents a direct measure of ISC proliferation. We used the temperature-sensitive driver escargot-gal4, UAS-GFP; tubulin-gal80ts (esgts>gfp; Micchelli & Perrimon, 2006) to induce over-expression of Src within ISCs/EBs (stem/progenitor cells) in the adult Drosophila midgut. The ectopic activation of Src kinase was achieved by overexpression of independent UAS-Src transgenes—wild-type Src64B (esgts>Src64WT) and constitutively active form of Src42A (esgts>Src42CA)—or by overexpression of an RNA interference transgene to knockdown Csk (Vidal et al, 2006; esgts>Csk-IR). All three approaches resulted in substantial ISC hyperproliferation and hyperplasia of the adult Drosophila intestine (Fig 1G and H and Supplementary Fig S1). Histological analysis of sections of paraffin-embedded midguts from 7-day-old esgts>Src64WT animals revealed ‘polyp-like’ structures containing multiple big and small BrdU+ve cell nuclei, which were not observed in age-matched control intestines (Fig 1I and I' and Supplementary Fig S1A and B). To further characterize the phenotype of esgts>Src64WT midguts, we stained tissues with anti-pH3 (Fig 1J–K') and anti-Delta (Fig 1M–O) antibodies to specifically label cells undergoing active mitosis and ISCs, respectively. Src–overexpressing midguts showed significant upregulation in the number of pH3+ve and Delta+ve cells when compared with control counterparts (Fig 1J–P and Supplementary Fig S1C and C'). Consistent with previous reports (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006), pH3+ve staining was restricted to small nuclei ISCs (Fig 1J–K') indicating that big BrdU+ve cell nuclei (Fig 1I') are likely to represent newly made, endoreplicating enterocytes (ECs; Micchelli & Perrimon, 2006). Importantly, Src-dependent ISC hyperproliferation in the midgut was largely suppressed by concomitant overexpression of the human homologue of Csk, Chk (Vidal et al, 2006; Fig 1L and Supplementary Fig S1G–J), highlighting the conserved nature of SFK regulation by upstream inactivating kinases. Altogether, these data demonstrate that enhanced expression of Src in stem/progenitor cells is sufficient to drive hyperproliferation and increase numbers of ISCs in the adult Drosophila midgut. Src is required to drive stem cell proliferation during intestinal homeostasis and regeneration We next tested whether endogenous Src was required for proliferation within the Drosophila intestine. To do this, we examined the role of Src in homeostatic self-renewal and damage-induced regeneration of the adult midgut. Like its vertebrate counterpart, the adult Drosophila posterior midgut is replenished by dedicated ISCs and displays a remarkable regenerative response to damaging agents (Cordero & Sansom, 2012). Damage-induced intestinal regeneration is characterized by an acute expansion of the stem/progenitor cell population and increase in ISC proliferation, which is required to regenerate the damaged intestinal epithleium (Amcheslavsky et al, 2009; Buchon et al, 2009; Jiang et al, 2009; Fig 2A and B). We first assessed the regulation of Src during intestinal regeneration after feeding flies with the pathogenic bacteria Pseudomonas entomophila (Pe; Buchon et al, 2009; Fig 2A–C). While immunostaining for pSrc showed barely detectable signal in the midgut epithelium from unchallenged animals, pSrc levels became more evident within the intestinal epithelium of animals subject to damage by Pe (Fig 2A–B'). RT–qPCR from whole midguts confirmed a threefold transcriptional upregulation of Src42 in regenerating tissues (Fig 2C). We next tested the functional role of Src42 during intestinal regeneration. We used RNAi transgenes to knockdown Src42 within stem/progenitors cells of the adult midgut (esgts>Src42-IR). Knockdown of Src42 by using two independent RNAi lines resulted in almost complete suppression of ISC proliferation in regenerating midguts (Fig 2D–F and Supplementary Fig S2). Midguts from animals heterozygous for a loss-of-function allele of Src42 (Src42K10108) also showed significantly impaired regeneration (Fig 2F and Supplementary Fig S2C and C') showing that Src42 is rate-limiting during regeneration. RNAi knockdown of Src64 did not significantly affect intestinal regeneration (data not shown). Figure 2. Src is required for ISC proliferation during homeostasis and regeneration of the adult Drosophila midgut A–B'. Immunofluorescence staining to detect pSrc (red) in midguts from esg > gfp (green) animals after feeding with Sucrose (Suc) (A, A') or subject to intestinal damage by feeding bacteria (Pe) (B, B'). Arrows point to examples of cell membranes stained with anti-pSrc. Scale bars, 20 µm. C. qRT-PCR from whole midguts as in (A–B') to detect transcript levels of Drosophila Src42 and Src64. Only Src42 was significantly upregulated in damaged midguts. Data represents average values ± SEM. D–E'. Posterior midguts from 14-day-old Suc- or Pe-fed control animals (D, D'; esgts > gfp) or animals subject to RNAi knockdown of Src42 in ISCs/EBs (E, E'; esgts > Src42-IR). F. Quantification of ISC proliferation in regenerating posterior midguts from animals of the indicated genotypes and treated as in (D–E'). Data represent average values ± SEM (***P < 0.0001 one-way ANOVA with Bonferroni's multiple comparison test). G–L. Homeostatic self-renewal in control and Src42-IR posterior midguts using the escargot ‘flip out’ system (esgts F/O). The lineage from gfp (control) and Src42-IR esg+ve cells (green) was analysed 7, 14 and 30 days after transgene induction. Scale bars, 50 µm. M–R. Adult posterior midguts carrying 7-, 14- and 30-day-old MARCM clones (green) from a control transgene (M–O; LacZ) or from Src RNAi (P–R; Src42-IR). Scale bars, 20 µm. S. Quantification of the number of cells per clone in posterior midguts as in (M–R). Note that, unlike their control counterparts, MARCM Src42-IR clones failed to grow and even decreased in size over time. Clonal size distribution is presented as a dot plot with the mean clonal size ± SEM (***P < 0.0001; **P < 0.001 one-way ANOVA with Bonferroni's multiple comparison test). Download figure Download PowerPoint We also noted that esgts>Src42-IR midguts looked ‘thinner’ than their control counterparts (Fig 2E; compare with Fig 2D and Supplementary Fig S2B; compare with Supplementary Fig S2A), even when unchallenged. We therefore tested whether Src42 was required for ISC proliferation during homeostatic self-renewal of the adult midgut. We first overexpressed a Src42-IR transgene within the intestinal epithelium using the inducible ‘escargot flip out’ system (esgts F/O>gfp; Jiang et al, 2009), in which every progenitor cell and its new progeny will express Gal4 and UAS-gfp in addition to the UAS-Src42-IR RNAi transgene. We then visualized the newly produced esg cell lineage 7, 14 and 30 days after transgene induction (Fig 2G–L). Knockdown of Src42 from the epithelium of undamaged midguts impaired homeostatic self-renewal in the adult posterior midgut (Fig 2G–L) as evidenced by the reduced esg linage labelling in esgts F/O>Src42-IR midguts after 14 and 30 days of tracing (Fig 2K and L; compare with Fig 2H and I). We next used the MARCM system (Lee & Luo, 1999) to create adult midgut clones from a control transgene (MARCM LacZ; Fig 2M–O) or with Src42 knockdown (MARCM Src42-IR; Fig 2P–R) and follow their growth at 7, 14 and 30 days after clonal induction (ACI). No significant difference was observed in the size of control and Src42-IR during the intimal phase of clonal growth (Fig 2M, P and S). However, unlike control clones, 14- and 30-day-old Src42-IR clones failed to progressively grow over time and even decreased their size (Fig 2Q–S; compare with 2N, O and S). These results indicate that endogenous Src42 activity is essential to sustain ISC proliferation during homeostatic self-renewal as well as to drive ISCs during regeneration of the adult Drosophila midgut. Src is activated downstream of Wg/Wnt signalling in the adult Drosophila midgut c-Src is hyperactivated in response to Apc loss in mouse and human intestinal adenomas (Fig 1C–F). Work from us and others has shown that Wnt/Wg signalling drives ISC hyperproliferation and is required for regeneration of the adult Drosophila midgut (Lin et al, 2008; Lee et al, 2009; Cordero et al, 2012a,b). We therefore tested whether Src activation was dependent on Wnt signalling in the midgut. Src42 mRNA showed a 1.7-fold upregulation in whole midguts from animals homozygous for the Apc1 null allele Apc1Q8 (Ahmed et al, 1998; Fig 3A). Immunofluorescence staining revealed pSrc upregulation within stem/progenitor cells from Apc1Q8 midguts (Fig 3B–C') and in midguts overexpressing Wg (esgts>wg) or an activated from of β–catenin/Armadillo (esgts>armS10; Fig 1D–F'). Importantly, upregulation of pSrc upon Pe infection was impaired in midguts unable to regenerate due to wg knockdown in stem/progenitor cells (esgts>wg-IR; Fig 3G–I'). Altogether these results suggest that Wnt signalling is necessary and sufficient for Src activation in the Drosophila midgut. Figure 3. Src is activated downstream of Wnt signalling in the adult Drosophila midgut A. qRT-PCR of whole midguts from 7-day-old control and Apc1−/− (Apc1Q8) animals to assess transcript levels of Src42 and Src64. Only Src42 was significantly upregulated in the midgut in response to Apc1 loss. Data represent average values ± SEM. B–C'. 7-day-old control (B, B') and Apc1Q8 midguts (C, C') co-stained with anti-pSrc (red) and anti-Delta (green). Apc1Q8 midguts display significant pSrc upregulation within Delta+ve ISCs (arrows). Scale bars, 20 µm. D–F'. pSrc immunofluorescence (red) in 14-day-old midguts from control animals or animals subjected to Wg signalling upregulation by overexpressing Wg or activated β-catenin/Armadillo within ISCs/EBs (esgts > gfp; esgts > wg and esgts > armS10, respectively). Note pSrc upregulation in ISCs/EBs in response to Wg signalling activation (arrows). Scale bars, 20 µm. G–I'. pSrc staining (red) in 14-day-old midguts from control animals or animals subject to wg knockdown by overexpressing a wg RNAi within ISCs/EBs (esgts>wg-IR) and fed with Suc (G, G') or Pe (H-I'). Note that pSrc upregulation in ISCs/EBs upon Pe feeding (H, H'; arrows) is suppressed by wg knockdown (I, I'). Scale bars, 20 µm. Download figure Download PowerPoint Src is required for tumourigenesis after Apc loss in Drosophila We next tested the functional role of Src42 in ISC hyperproliferation driven by loss of Apc1 in the adult Drosophila midgut (Cordero et al, 2012a). We created adult midgut clones of cells deficient for Apc1 only (MARCM Apc1Q8; Fig 4B) or combined with Src42 knockdown (MARCM Src42-IR; Apc1Q8; Fig 4D). As previously reported, posterior midgut Apc1−/− mutant clones contained significantly more cells and displayed increased ISC proliferation when compared with control (MARCM Lac-Z) clones (Cordero et al, 2012a; Fig 4A, B, E and F). Consistent with our previous results (Fig 2M–S), Src42-IR clones (MARCM Src42-IR) were smaller than control clones (Fig 4A, C, E and F). Importantly, knocking down Src42 inhibited hyperproliferation in Apc1−/− clones (Fig 4B, D, E and F). These data demonstrate an essential role for Src42 in Apc1-dependent ISC hyperproliferation in the adult Drosophila midgut. Figure 4. Src mediates Apc1-dependent intestinal hyperproliferation in the Drosophila midgut A–D. Adult posterior midguts carrying 14-day-old mARCM clones (green) of the indicated genotypes. Scale bars, 20 µm. E, F. Quantification of the number of cells per clone (E) and percentage of pH3+ve clones (F) in posterior midguts as in (A–D). Note that Src42 knockdown completely suppressed ISC proliferation in Apc1Q8 MARCM clones (B, D, E, F). Clonal size distribution is presented as a dot plot with the mean clonal size ± SEM (***P < 0.0001 one-way ANOVA with Bonferroni's multiple comparison test). Download figure Download PowerPoint Src drives ISC hyperproliferation through regulation of the EGFR/MAPK and Stat signalling pathways The EGFR/Ras/MAPK and Jak/Stat signalling pathways are required for Drosophila intestinal homeostasis and regeneration (Buchon et al, 2009; Jiang et al, 2009, 2011; Beebe et al, 2010; Biteau & Jasper, 2011) and are essential mediators of Apc1-driven intestinal hyperproliferation (Cordero et al, 2012a). We therefore tested whether these pathways were potential mediators of the role of Src in ISC proliferation in vivo. Src overexpression (esgts>Src64WT and esgts>Src42CA) resulted in ectopic activation of MAPK/Erk1/2 (pErk1/2; Fig 5A, A', C, C', E-G and not shown). Such phenotype resembled the one resulting from overexpression of activated Ras in stem/progenitor cells (Jiang et al, 2011) and correlated with upregulation of total levels of EGFR within the same cells (Fig 5B, B', D and D' and not shown). Transcriptional upregulation of egfr by Src overexpression was confirmed through qRT–PCR from whole midguts (Fig 5H). Importantly, knocking down EGFR or Ras completely suppressed ISC hyperproliferation in esgts>Src midguts (Fig 5J–L and Supplementary Fig S3A-I). Altogether, our results suggest that EGFR expression and downstream pErk activation are key mediators of intestinal hyperproliferation driven by Src. Figure 5. Src drives ISC hyperproliferation through upregulation of EGFR/MAPK and Stat signalling A-D'. Immunofluorescence from control (A–B'; esgts > gfp) and Src-overexpressing midguts (C–D'; esgts > Src64WT) stained with anti-pErk1/2 (A, A', C, C'; red or grey) or anti-EGFR (B, B', D, D'; red or grey) after 7 days of transgene expression. Src overexpression in ISCs/EBs results in significant upregulation of EGFR and ectopic pErk1/2. Scale bars: 20 µm. E-F'. Magnified views from midguts as in (A, A', C, C'). Note that, while in control midguts pErk1/2 staining is restricted to small nuclei esg > gfp+ve cells (E, E'; arrowhead), esgts > Src64WT midguts show ectopic pErk1/2 staining in big nuclei esg > gfp+ve cells (F, F'; arrows). G. Quantification of the esg > gfp/pErk1/2 cell area in midguts as in (E–F'). Data represent mean values ± SEM (***P < 0.0001 unpaired t-test). H, I. qRT-PCR from whole midguts of genotypes as in (A–D') to measure degfr transcript levels (H) and Stat signalling activation thorough Socs36E levels (I). Src overexpression results in transcriptional upregulation of egfr and Socs36E in the midgut. Data represent average values ± SEM. J-M. Quantification of ISC proliferation in posterior midguts from animals of the indicated genotypes after 7 days of transgene expression. Knockdown of EGFR/Ras in ISC/EBs suppresses hyperproliferation in Src-overe" @default.
• W2021181797 created "2016-06-24" @default.
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• W2021181797 date "2014-04-30" @default.
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• W2021181797 title "c-Src drives intestinal regeneration and transformation" @default.
• W2021181797 cites W1502391354 @default.
• W2021181797 cites W1594706458 @default.
• W2021181797 cites W1963570775 @default.
• W2021181797 cites W1964684127 @default.
• W2021181797 cites W1965157206 @default.
• W2021181797 cites W1968112233 @default.
• W2021181797 cites W1968248547 @default.
• W2021181797 cites W1976598331 @default.
• W2021181797 cites W1977455794 @default.
• W2021181797 cites W1985234259 @default.
• W2021181797 cites W1989388426 @default.
• W2021181797 cites W1992873952 @default.
• W2021181797 cites W1993081168 @default.
• W2021181797 cites W1994216131 @default.
• W2021181797 cites W1999563094 @default.
• W2021181797 cites W2006229889 @default.
• W2021181797 cites W2011623297 @default.
• W2021181797 cites W2026901159 @default.
• W2021181797 cites W2029556699 @default.
• W2021181797 cites W2030565228 @default.
• W2021181797 cites W2031429066 @default.
• W2021181797 cites W2032674547 @default.
• W2021181797 cites W2033728878 @default.
• W2021181797 cites W2034385303 @default.
• W2021181797 cites W2036988959 @default.
• W2021181797 cites W2047482319 @default.
• W2021181797 cites W2051909115 @default.
• W2021181797 cites W2052203880 @default.
• W2021181797 cites W2059813946 @default.
• W2021181797 cites W2065653828 @default.
• W2021181797 cites W2070386387 @default.
• W2021181797 cites W2073609428 @default.
• W2021181797 cites W2075773348 @default.
• W2021181797 cites W2080506450 @default.
• W2021181797 cites W2080574458 @default.
• W2021181797 cites W2081495016 @default.
• W2021181797 cites W2085423858 @default.
• W2021181797 cites W2090398095 @default.
• W2021181797 cites W2090562908 @default.
• W2021181797 cites W2098253798 @default.
• W2021181797 cites W2104005887 @default.
• W2021181797 cites W2105575647 @default.
• W2021181797 cites W2106918615 @default.
• W2021181797 cites W2111130664 @default.
• W2021181797 cites W2134359750 @default.
• W2021181797 cites W2135055828 @default.
• W2021181797 cites W2135715423 @default.
• W2021181797 cites W2142018200 @default.
• W2021181797 cites W2143974518 @default.
• W2021181797 cites W2146167376 @default.
• W2021181797 cites W2155255760 @default.
• W2021181797 cites W2157010562 @default.
• W2021181797 cites W2164193456 @default.
• W2021181797 cites W2165885715 @default.
• W2021181797 cites W2166394159 @default.
• W2021181797 cites W2171472978 @default.
• W2021181797 cites W2262414037 @default.
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