MAPK Signaling Regulates the Proliferation of Drosophila Renal and Nephric Stem Cells

Available online at www.sciencedirect.com

ScienceDirect Journal of Genetics and Genomics 42 (2015) 9e20

JGG ORIGINAL RESEARCH

EGFR/MAPK Signaling Regulates the Proliferation of Drosophila Renal and Nephric Stem Cells Zhouhua Li a,b,*, Sen Liu b, Yu Cai b,c,* a

College of Life Sciences, Capital Normal University, Beijing 100048, China Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore c Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore b

Received 18 June 2014; revised 26 November 2014; accepted 29 November 2014 Available online 9 December 2014

ABSTRACT Tissue homeostasis, accomplished through the self-renewal and differentiation of resident stem cells, is critical for the maintenance of adult tissues throughout an animal’s lifetime. Adult Drosophila Malpighian tubules (MTs or fly kidney) are maintained by renal and nephric stem cells (RNSCs) via self-renewing divisions, however, it is unclear how RNSC proliferation and differentiation are regulated. Here we show that EGFR/MAPK signaling is dispensable for RNSC maintenance, but required for RNSC proliferation in vivo. Inactivation of the EGFR/MAPK pathway blocks or greatly retards RNSC cell cycle progression; conversely, over-activation of EGFR/MAPK signaling results in RNSC over-proliferation and disrupts the normal differentiation of renablasts (RBs), the immediate daughters of RNSC divisions. Our data further suggest that EGFR/MAPK signaling functions independently of JAK/STAT signaling and that dMyc and CycE partially mediate EGFR/MAPK signaling in MTs. Together, our data suggest a principal role of EGFR/MAPK signaling in regulating RNSC proliferation, which may provide important clues for understanding mammalian kidney repair and regeneration following injury. KEYWORDS: Drosophila Malpighian tubules; Renal and nephric stem cells; EGFR/MAPK signaling

INTRODUCTION Stem cells are a group of cells with unique potential, which undergo asymmetric divisions to self-renew and produce differentiating daughters at the same time. The maintenance of adult tissue homeostasis, during which out-going differentiated cells are replenished by differentiating progeny of resident stem or progenitor cells, is essential for life. Growing evidence suggests that stem cell self-renewal and

Abbreviations: EGFR, epidermal growth factor receptor; JAK/STAT, Janus kinase/signal transducers and activators of transcription; MAPK, mitogenactivated protein kinase; MTs, Malpighian tubules; RNSC, renal and nephric stem cell. * Corresponding authors. Tel: þ86 10 6890 1531 (Z. Li); Tel: þ65 6872 7419, fax: þ65 6872 7000 (Y. Cai). E-mail addresses: [email protected] (Z. Li); [email protected] (Y. Cai).

differentiation are controlled by both intrinsic factors and extrinsic signals (Morrison and Spradling, 2008). Deregulation of stem cell self-renewal versus differentiation could result in depletion or excessive proliferation of stem cells, which eventually leads to premature aging or cancer. Thus, understanding the molecular mechanisms governing stem cell self-renewal versus differentiation is crucial for the use of stem cells in regenerative medicine and cell therapy. The function of excretory systems (such as kidney in vertebrates and Malpighian tubules (MTs) in Drosophila) is important for adult homeostasis by removing metabolic wastes, foreign toxins and maintaining ionic, acid/base and water balance (Dow and Davies, 2006). It is well known that the mammalian kidney has a great potential for tissue regeneration following an ischemic or toxic injury. Ischemic injury to the mammalian kidney causes acute renal failure, loss of tubular polarity, necrosis and cell death, followed by tubular

http://dx.doi.org/10.1016/j.jgg.2014.11.007 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

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regeneration and recovery of renal function (Anglani et al., 2008; Gupta and Rosenberg, 2008; Vaidya et al., 2008; Yokoo et al., 2008). Furthermore, many animal models provide evidence of regeneration of completely degenerated renal tissues after injury. These data strongly suggest the existence of adult kidney stem cells and that a stem cell-based system may function during the regeneration processes in vertebrates (Elger et al., 2003; Haller et al., 2005; Anglani et al., 2008). However, these hypotheses await further experimental supports. Drosophila MTs show great similarities in development and function to the mammalian kidney (Dow and Davies, 2006; Singh et al., 2007; Singh and Hou, 2009). Even some of the pathways and molecules utilized during development are conserved in both systems (Ainsworth et al., 2000; Denholm et al., 2003; Jung et al., 2005). Adult Drosophila has two pairs of MTs, a longer anterior pair that runs through the hemolymph on both sides of the midgut and a shorter posterior pair that runs along the hindgut, converging through common ureters onto the alimentary canal at the midguthindgut junction (Sozen et al., 1997; Pugacheva and Mamon, 2003; Singh et al., 2007). Genetic mapping revealed that each tubule can be divided into four compartments with six regions including initial segment, transitional segment, main segment, lower tubules, upper ureter and lower ureter (Sozen et al., 1997). Following its formation during late embryogenesis Drosophila MTs are thought to be stably maintained throughout development. However, using lineage tracing and molecular marker labeling, it was shown that Drosophila MTs actually contain multipotent stem cells (known as renal and nephric stem cells, RNSCs), which are located at the lower tubule and ureter region and produce several differentiated cell types. Moreover, autocrine JAK/STAT signaling was shown to regulate RNSC self-renewal (Singh et al., 2007). Both RNSCs and RBs (the differentiating daughters of RNSC divisions) are small diploid cells expressing STAT92E, Armadillo (Arm, the fly b-Catenin) and DE-Cadherin (DE-Cad). However, it is likely that only RNSCs express Upd, a ligand of the JAK/ STAT pathway, and undergo cell cycle progression (Singh et al., 2007). Whether RNSCs are regulated by additional signaling pathways remains elusive. The epidermal growth factor receptor (EGFR) pathway is widely utilized during animal development and is involved in cell fate specification (Shilo, 2003). In Drosophila, the signaling cascade is initiated upon binding of ligand (Spitz, Gurken, Keren and Vein in Drosophila) to the receptor and acts through the canonical RAS/RAF/MEK/mitogen-activated protein kinase (MAPK) pathway and the ETS transcriptional activator, Pointed (Pnt), to regulate gene expression (Shilo, 2003). Mis-regulation of this pathway is often associated with developmental defects including a variety of cancer (Holbro and Hynes, 2004). Previous data indicate that this pathway is also required for the normal development of stem cells under physiological conditions (Aguirre et al., 2010). In Drosophila midgut, EGFR/MAPK signaling acts to maintain midgut homeostasis under physiological conditions and mediate regenerative response under stress conditions (Buchon

et al., 2010; Jiang et al., 2010; Biteau and Jasper, 2011; Xu et al., 2011). Whether EGFR/MAKP signaling is required for RNSC proliferation is unknown. Here, we show that EGFR/MAPK signaling functions in Drosophila MTs to maintain tissue homeostasis. Our data suggest that the EGFR/MAPK pathway is likely not essential for RNSC maintenance, but is critical for RNSC proliferation and RB differentiation. Our data indicate that its role in RNSCs is independent to that of JAK/STAT signaling and identify dMyc and CycE as two downstream mediators of EGFR/MAPK signaling. Given that the regulation of EGFR/ MAPK signaling is evolutionarily conserved between flies and mammals, our study may shed light on the mechanisms underlining renal regeneration after ischemic injury in mammals. RESULTS EGFR/MAPK signaling is activated in RNSCs and RBs We first addressed whether EGFR/MAPK pathway components are expressed in adult Drosophila MTs. In wild type (WT), RNSCs are small diploid cells located in lower tubules and ureters and undergo self-renewing divisions to generate a new-born RNSC daughter and an RB daughter, which differentiates directly into a renalcyte (RC) in the region of the lower tubules and ureters, or a type I or II cell in the region of upper tubules. Using an anti-EGFR antibody in immunofluorescence analysis, we showed that EGFR was expressed in MTs and interestingly only in the small cells located in low tubules and ureters, a region with high stem cell activity (Fig. 1A). To investigate whether these EGFR-expressing cells are RNSCs and/or RBs, we co-stained MTs with both antiEGFR and anti-STAT92E antibodies. Our results showed that all EGFR-expressing cells were also STAT92E-positive, indicating an RNSC/RB fate (Fig. 1A). In Drosophila, the EGFR pathway can signal through the canonical RAS/RAF/ MEK/MAPK pathway to regulate target gene expression via the Pointed (Pnt) transcriptional activator (Shilo, 2005). PntlacZ, an enhancer trap line for Pnt transcription activation, is specifically expressed in the small cells located in the region of lower tubules and ureters which also express STAT92E (Fig. 1B). To further address whether the EGFR/MAPK pathway is activated in MTs, we used an antibody specific for the active, double phosphorylated form of ERK (pERK), representing signal activation in vivo in immunofluorescence analysis (Gabay et al., 1997). Indeed, the pERK positive cells were small cells situated in the region of lower tubules and ureters and also STAT92E-positive (Fig. 1C). In adult Drosophila midgut, the Notch pathway is activated and Delta (Dl), one of the Notch ligands in the fly, serves as an intestinal stem cell (ISC)-specific marker (Ohlstein and Spradling, 2007). Our recent data show that the Notch pathway is activated in adult MTs and Dl serves as an RNSC-specific marker (Li et al., 2014). Interestingly, pERK signal was highly detected in these Dl-positive cells (data not shown). Together, these data show that components of the EGFR/MAPK

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Fig.1. EGFR/MAPK signaling pathway is activated in MTs. A: EGFR (red, shown in panel A0 ) is detected in the STAT92E-positive cells (green, shown in panel A00 ) in wild-type (WT) MTs (white arrowheads). B: Pointed (measured by Pnt-lacZ, red, shown in panel B0 ) expresses in the STAT92E-positive cells (green, shown in panel B00 ) in WT MTs (white arrowheads). C: EGFR signaling is activated (measured by pERK, red, shown in panel C0 ) in the STAT92E-positive cells (green, shown in panel C00 ) in WT MTs (white arrowheads). DNA in blue. Scale bars: 10 mm.

pathway are expressed and this signaling pathway is activated in RNSCs and RBs. EGFR/MAPK signaling controls RNSC proliferation To determine whether EGFR/MAPK signaling functions in RNSCs, we used the mosaic analysis with a repressible cell marker (MARCM) system to generate raf11, an raf amorphic allele, mutant clones (Lee and Luo, 1999). It has been shown that RNSCs have a slow proliferation rate compared to ISCs (Singh et al., 2007). Under our culture conditions the GFPpositive RNSCs roughly divided once per week. In WT control flies, GFP-positive clones could grow up to 3e4 cells within 28 days after clonal induction (ACI) (Fig. 2A and B). However, under the same conditions Raf11 mutant clones consisted of single small cell expressing the RNSC markers, STAT92E and Dl, indicating an RNSC/RB fate (Fig. 2C and data not shown). We also deployed a Flp-out system (AY-Gal4 system) (Ito et al., 1997), in combination with the RNAi technique, to knockdown various components of the EGFR/ MAPK pathway including EGFR, Ras, Raf and Pnt. In controls, GFP-positive clones could grow up to 2e3 cells at 28 days ACI (Fig. 2D and E), consistent with our MARCM analyses. However, clones with compromised EGFR signaling mostly consisted of a single cell that maintained STAT92E expression (Fig. 2F). Furthermore, when a dominant-negative form of EGFR, EGFRDN, was induced in MTs, all clones contained only a single cell 30 days ACI (Fig. 2G). Similar phenotypes were observed in clones with compromised Ras, Raf or Pnt activity (Fig. 2H and Fig. S1). It was shown that the

transmembrane protein Kekkon-1 forms a negative feedback loop to down-regulate EGFR signaling and ectopic expression of Kekkon-1 mimics loss of EGFR activity (Ghiglione et al., 1999). When kekkon-1 was ectopically expressed in MTs using the AY-Gal4 system (Ito et al., 1997), almost all kekkon1-expressing clones contained a single cell with STAT92E and Dl expression 23 days and 40 days ACI, respectively, while the WT control clones containing up to 4 cells can be observed 40 days ACI (Fig. 2I and J). These data show that EGFR/ MAPK signaling is not required for RNSC maintenance under our experimental conditions, but is essential for RNSC proliferation. We next addressed whether ectopic EGFR signaling in RNSCs could result in over-proliferation of renal cells using the AY-Gal4 system in conjunction with UAS transgenes (Ito et al., 1997). Compared to control clones which contained 2e3 cells 28 days ACI (Fig. 3A and B), clones expressing ltop, a constitutively active form of EGFR (Queenan et al., 1997; Duchek and Rorth, 2001), led to the formation of large clusters with small cells, resembling RNSCs/RBs. Typically, these clones grew up to about 2e6 cells within 3 days ACI and eventually ended up with massive overproliferation (Fig. 3C and D). To confirm this, we stained MTs with phospho-Histone H3 (PH3), a mitotic marker. In controls, roughly only 2 PH3-positive cells could be identified in every 5 pairs of MTs (5 flies), indicating a low proliferation rate. However, each MTs expressing letop exhibited multiple PH3-positive cells, indicating that ectopic letop expression caused ectopic proliferation of renal cells (Fig. 3E). Similar but stronger over-proliferation of RNSCs was observed when a

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Fig. 3. Over-activation of EGFR/MAPK signaling in RNSCs causes over-growth of renal cells. A: A control Flp-out clone (white arrowhead, GFP, green, shown in panel A0 in blackwhite) contains a single cell 5 days ACI. B: A control Flp-out clone (white arrowhead, GFP, green, shown in panel B0 in blackwhite) contains 4 cells 28 days ACI (STAT92E in red, DNA in blue). C: A clone expressing letop (white arrowhead, GFP, green, shown in panel C0 in blackwhite), a constitutively activated form of EGFR, contains multiple STAT92E-positive cells (red) 5 days ACI. D: Quantification of clone size of ectopic letop expression 3 days ACI. E: Quantification of PH3-positive cells in control and letop expressing MTs 6 days ACI (30 flies counted for each sample). F: Quantification of clone size (cell number) of control and Rafgof clones 1 day ACI. G: A Rafgof expressing clone (white arrowhead, GFP, green) 3 days ACI contains several Arm-positive cells (red, shown in panel G0 in blackwhite) (DNA in blue). H: Ectopic expression of Rafgof (white arrowhead, GFP, green, shown in panel H0 in blackwhite) 5 days ACI generates tumorous clones (DNA in blue). I: A pair of control MTs of Ay > Rafgof without heat shock. I0 : Close-up view of the dashed region in I, showing the junction between the upper ureters and the low tubules. J: A pair of MTs expressing Rafgof, note the enlarged size of the lower tubule and ureters regions. J0 : Close-up view of the dashed region in J, showing the massive cell clusters indicated (black arrowheads). K: A clone expressing PntP1 (white arrowhead, GFP, green), a constitutively active form of Pnt, contains several STAT92E-positive cells (red) 6 days ACI. L: A clone with compromised Cbl activity (cblRNAi, cbli, white arrowhead, GFP, green) contains multiple STAT92E-positive cells (red) 5 days ACI. DNA in blue in panels except graphs. Scale bars: 10 mm (AeC, G, H, K and L); 5 mm (I and J); 1 mm (I0 and J0 ).

Fig. 2. EGFR/MAPK signaling pathway is required for RNSC proliferation. A: A control MARCM clone (white arrowhead, GFP, green, shown in panel A0 ) contains single cell expressing STAT92E (red) 5 days after clone induction (ACI). B: A control MARCM clone (white arrowhead, GFP, green, shown in panel B0 ) comprises of 3 cells 28 days ACI (STAT92E in red). C: A Raf11 mutant MARCM clone (white arrowhead, GFP, green, shown in panel C0 ) contains single STAT92E-positive (red) cell 28 days ACI, suggesting defective proliferation of Raf mutant clone. D: A control Flp-out clone (white arrowhead, GFP, green, shown in panel D0 ) contains single cell 5 days ACI. E: A control Flp-out clone (white arrowhead, GFP, green, shown in panel E0 ) comprises of 4 cells 28 days ACI (STAT92E in red). F: Knock-down of EGFR (EgfrRNAi, Egfri) results in clones (white arrowheads, green, shown in panel F0 ) containing a single STAT92E-positive cell (red) 30 days ACI. G: Ectopic EgfrDN expression results in clones (white arrowhead, green, shown in panel G0 ) containing only a single STAT92E-positive cell (red) 30 days ACI. H: Knock-down of Ras (Rasi) also results in clones (white arrowhead, green, shown in panel H0 ) containing only a single STAT92E-positive cell (red) 30 days ACI. I: Ectopic expression of kekkon-1, results in clones (white arrowhead, green, shown in panel I0 ) containing only a single STAT92E-positive cell (red) 23 days ACI. J: Quantification of clone size (cell number/clone) of WT control and kekkon1 over-expressing clones. GFP in green, DNA in blue (D, G and H), LamC in blue (E). Scale bars: 10 mm.

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Fig. 4. Ectopic EGFR/MAPK signaling disrupts RB differentiation. A: A control clone remains as a single cell (white arrowhead) which expresses Arm (red, shown in panel A0 ) 5 days ACI. B: A clone expressing Rafgof contains multiple Arm-positive small cells (white arrowhead, red, shown in panel B0 ) 3 days ACI. C: A control clone remains as a single cell with STAT92E expression (red, shown in panel C0 , white arrowhead) 4 days ACI. D: A Rafgofeexpressing clone contains multiple STAT92E-positive cells (red, shown in panel D0 ) (white arrowheads) 4 days ACI. E: upd-lacZ (red, shown in panel E0 in blackwhite) is weakly detected in WT RNSCs (white arrowhead). F: Multiple upd-lacZ positive cells (red, shown in panel F0 in blackwhite) are observed in those Rafgof expressing clones 3 days ACI (white arrowheads). G: A control single cell clone labeled by PH3 (white arrowhead, red, shown in panel G0 in blackwhite), the mitotic marker. H: Multiple PH3-positive cells (red, shown in panel H0 in blackwhite) are observed in those clones expressing Rafgof 4 days ACI (white arrowheads). DNA in blue, clones labeled by GFP in green. Scale bars: 10 mm.

constitutively active form of Raf (Rafgof) was introduced in MTs (Brand and Perrimon, 1994). In WT control clones, essentially all GFP-marked clones contained only one small cell 5 days ACI (Fig. 3A); however, clones over-expressing Rafgof formed a cluster of small cells only 1 day ACI (Fig. 3F), and these clusters continued to grow and formed a tumorous mass 5 days ACI (Fig. 3G and H). Eventually, these MTs exhibited hyperplasia morphology (compare Fig. 3I and J). Similarly, ectopic PntP1 expression, an active form of Pnt (Klaes et al., 1994), also led to renal cell over-proliferation (Fig. 3K). Finally, compromising Cbl, a negative regulator of EGFR signaling (Pai et al., 2000), in MTs also caused RNSC over-proliferation in the region of low tubules and ureters with clones filled with small cells (Fig. 3L). Taken together, these data demonstrate that ectopic EGFR/MAPK signaling in MTs results in over-proliferation of renal cells. Ectopic EGFR/MAPK signaling disrupts RB differentiation Although autocrine JAK/STAT signaling maintains RNSC fate, ectopic JAK/STAT signaling does not disrupt RB differentiation as normal lineages were observed in clones with ectopic JAK expression (Singh et al., 2007) (data not shown). Interestingly, we noticed that most cells in clones with ectopic EGFR/MAPK signaling were small cells, likely RNSCs and/or

RBs, a phenotype distinct from clones with ectopic JAK/STAT signal activation (Fig. 3C, G, H, K, L and Fig. S2). We then looked at the identity of these small cells generated by ectopic EGFR/MAPK signaling in detail, focusing on Rafgof overexpressing clones. First, we examined Armadillo (Arm, the fly b-catenin homolog) expression which strongly outlines the small cells including RNSCs and RBs. As reported previously, we found that in WT MTs Arm strongly outlined small cells including RNSCs and RBs (Fig. 4A). Interestingly, the small cells in Rafgof over-expressing clusters were strongly labeled by Arm, suggesting that these cells are probably RNSCs and/or RBs (Fig. 4B). Second, STAT92E was identified as a marker expressed in both RNSCs and RBs in WT MTs (Fig. 4C) (Singh et al., 2007). We found that most, if not all, cells in the Rafgof over-expressing clusters expressed STAT92E, confirming that these small cells are RNSCs and/or RBs but not RCs (Fig. 4D). Third, it has been shown that in WT MTs, JAK/ STAT signaling functions in an autocrine manner to maintain RNSCs and Upd-expression (measured by upd-Gal4) serves as an RNSC marker. Here, we examined Upd-expression using upd-lacZ, an upd transcription reporter (Chao et al., 2004). Although upd-lacZ was only weakly detected in WT RNSCs, several upd-lacZ positive cells were identified in each Rafgof over-expressing cluster, suggesting that multiple RNSCs existed in each cluster (Fig. 4E and F). We also found some

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Fig. 5. EGFR/MAPK signaling functions independently of JAK/STAT signaling. A: A control clone remains as a single cell (white arrowhead) expressing Arm (red, shown in panel A0 ) 5 days ACI. B: A STAT92E mutant clone (MARCM clone of STAT92Ej6c8) (white arrowhead, GFP, green, shown in panel B0 ) led to direct differentiation of RNSC into RC (arrowhead). C: Clones expressing Rafgof (GFP, green, shown in panel C0 ) contains multiple small Arm-positive cells (white arrowheads) 5 days ACI. D: Clones with Rafgof expression and defective in STAT92E function (by MARCM clone technique) (GFP, green, shown in panel D0 ) contain many small Arm-positive cells (white arrowheads), similar to Rafgof expressing clones (C). E: Compromising Hop activity (by RNAi, Hopi) (GFP, green, shown in panel E0 ) leads to the direct differentiation of RNSCs (white arrowhead). F: Clones expressing Rafgof but defects in Hop activity (HopRNAi) (white arrowheads, GFP, green, shown in panel F0 ) contained many small cells, similar to clones expressing Rafgof (C). G: Ectopic expression of Upd (GFP, green, shown in panel G0 ) resulted in the formation of ectopic small cells (white arrowheads) as well as large differentiated ECs (red arrowheads). H: Compromising Ras activity (by RNAi, Rasi) (GFP, green, shown in panel H0 ) blocks the proliferation of RNSCs (white arrowheads). I: Clones expressing upd but with defective Ras activity (RasRNAi, Rasi) (GFP, green, shown in panel I0 ) differentiate into large RCs (white arrowheads). J: RNSC proliferation is blocked in clones with defective Raf activity (RafRNAi, 10 days ACI) (GFP, green, shown in panel J0 ) (white arrowheads). K: Knock-down of STAT92E (STATRNAi, STATi), GFP, green, shown in panel K0 ) results in direct differentiation of RNSCs into RCs (white arrowheads) 7 days ACI. L: Clones simultaneously defective in STAT92E and Raf activity (STAT92ERNAi; RafRNAi) (GFP, green, shown in panel L0 ) differentiates into RC (white arrowhead) 7 days ACI. DNA in blue, clones are marked by GFP in green, Arm in red. Scale bars: 10 mm.

small cells with higher JAK/STAT signal activation than others, consistent with the proposed model that RNSCs show higher levels of JAK/STAT signaling than RBs (measured by 10  STAT:GFP, a STAT92E reporter-GFP in vivo, Fig. S3). Fourth, it was proposed that although RNSCs and RBs are both positive for STAT92E, only RNSCs can undergo cell cycle progression (Singh et al., 2007). In WT, all PH3-positive cells examined (n ¼ 20) are also STAT92E-positive, consistent with the notion that only RNSCs divide in vivo. In WT, PH3positive cells were rarely observed (roughly 2 cells in every 5

pairs of MTs). However, several PH3-positive cells could be observed in each Rafgof over-expressing cluster, further suggesting that multiple RNSCs exist in each cluster (Fig. 4G and H). Fifth, we recently documented that, similar to ISCs in the midgut, RNSCs express high levels of Dl which activates Notch signaling in RBs (measured by þ Su(H)-lacZ reporter expression) to induce differentiation (Li et al., 2014). In WT, each RNSC lineage contains only one Dl-positive RNSC. Several Dl-positive cells were observed in each Rafgof overexpressing cluster, indicating that ectopic EGFR/MAPK

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Fig. 6. EGFR/MAPK signaling regulates RNSC proliferation partially through dMyc and CycE. A: dMyc (red, shown in panel A0 ) is hardly detected in WT RNSCs/RBs (white arrowheads). B: dMyc (red, shown in panel B0 ) is elevated upon Rafgof overexpression (white arrowheads). C: CycE (red, shown in panel C0 ) is weakly expressed in a WT RNSC (white arrowhead). D: CycE (red, shown in panel D0 ) is upregulated upon Rafgof overexpression (white arrowhead). E: A control Flp-out clone exhibits one small cell with Arm expression (red, shown in panel E0 ) 5 days ACI (white arrowhead). F: A dMyc expressing clone (GFP, green, shown in panel F0 ) contains several small cells 5 days ACI (white arrowhead). G: A CycE expressing clone (GFP, green, shown in panel G0 ) contains several small cells 5 days ACI (white arrowhead). H: An RNSC clone compromised in dMyc function (by RNAi. dMyci) (GFP, green, shown in panel H0 ) contains a single cell 15 days ACI (white arrowheads), similar to compromising EGFR/MAPK function. I: RNSC proliferation is blocked in clones with defective CycE (CycERNAi) (GFP, green, shown in panel I0 ), resulting in one small cell 10 days ACI (white arrowheads). J: Ectopic Rafgof expression produces tumorous clones (GFP, green, shown in panel J0 ) 5 days ACI (white arrowheads). K: Compromising dMyc function (by dMycRNAi) in these Rafgof expressing clones (GFP, green, shown in panel K0 ) largely suppresses the phenotype 5 days ACI (white arrowhead). L: Ectopic Dap expression effectively suppresses the over-grow phenotype associated with ectopic Rafgof expression (GFP, green, shown in panel L0 ) 5 days ACI (white arrowhead). M: Reducing CycE activity strongly suppresses the over-growth phenotype induced by ectopic Rafgof expression (GFP, green, shown in panel M0 ) 5 days ACI (white arrowheads). N: Removal of CycE function in clones expressing Rafgof totally suppresses the over-growth phenotype (GFP, green, shown in panel N0 ) 5 days ACI (white arrowheads). O and P: Ectopic dMyc (O) and CycE (P) expression in clones with compromised Raf function (RafRNAi, Rafi), GFP, green, shown in panels O0 and P0 respectively) results in clones containing several small cells 5 days ACI, resembling dMyc and CycE expressing clones (white arrowheads). DNA in blue and GFP in green. Scale bars: 10 mm.

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signaling results in the formation of ectopic RNSCs. However, it is also worthy to note that these clusters also contain multiple Gbe þ Su(H)-lacZ-positive cells, indicative of an RB fate (Fig. S4). Similar phenotypes were obtained in clones expressing l-EGFR (data not shown). Collectively, these data demonstrate that over-activation of EGFR/MAPK signaling leads to RNSC over-proliferation and the generation of tumorous clusters containing both RNSCs and RBs. Furthermore, ectopic EGFR/MAPK signaling also disrupts RB differentiation.

observed in the Raf/STAT92E double mutant (Fig. 5L), supporting the notions that inactivation of EGFR/MAPK signaling does not permanently block renal cell differentiation, and that EGFR/MAPK signaling does not function downstream of JAK/STAT signaling. Taken together, these data suggest that the EGFR/MAPK and the JAK/STAT pathways likely function in parallel to regulate RNSC self-renewal and proliferation.

EGFR/MAPK signaling functions independently of JAK/STAT signaling

The aforementioned experiments demonstrate the essential roles of EGFR/MAPK signaling in regulating RNSC proliferation and differentiation. How does EGFR/MAPK signaling regulate the proliferation and differentiation of RNSCs? We further examined the mechanism(s) by which EGFR/MAPK signaling regulates RNSC proliferation and differentiation. Over-activation of EGFR/MAPK signaling resulted in overproliferation of RNSCs/RBs, suggesting an up-regulation of the cell cycle machinery. We explored this possibility and focused on dMyc and CycE, two well-studied cell cycle regulators. In WT, dMyc and CycE were hardly detected in MTs (Fig. 6A and C). However, both dMyc and CycE were highly up-regulated in clones expressing Rafgof (Fig. 6B and D), indicating that they might function downstream of EGFR/ MAPK signaling. Next, we addressed the roles of dMyc and CycE in RNSC proliferation and differentiation. First, overexpression of either dMyc or CycE stimulated the proliferation of RNSCs (Fig. 6EeG), similar to the MTs with ectopic EGFR signaling (Fig. 3C and F). However, the extent of overgrowth of these clones was significantly weaker than clones with ectopic EGFR signal activation. Inactivation of either dMyc or CycE strongly inhibited the proliferation of RNSCs, similar to those seen with EGFR/MAPK loss of function, supporting the idea that both dMyc and CycE are required for RNSC proliferation (Fig. 6H and I). To ascertain whether EGFR/MAPK signaling acts through dMyc and CycE, the following experiments were carried out. First, over-proliferation of renal cells resulted by ectopic EGFR/MAPK signaling could be strongly suppressed by the simultaneous removal of dMyc (Fig. 6J and K). These results indicate that EGFR/MAPK signaling mediated overgrowth of renal cells requires dMyc activity. Second, overexpression of Dacapo (p27) (Lane et al., 1996), the inhibitor of CycE, effectively suppressed the formation of these tumorous clusters in a Rafgof background, indicating that CycE may function downstream of EGFR/MAPK signalling (Fig. 6L). Consistently, removing one copy of CycE partially suppressed the formation of the tumorous clusters associated with ectopic Rafgof expression (Fig. 6M), while complete removal of CycE function totally suppressed the formation of these clusters and resulted in only single cell clones, in agreement with the idea that CycE functions downstream of EGFR/MAPK signaling to regulate RNSC proliferation (Fig. 6N). Furthermore, overexpression of dMyc or CycE can still drive ectopic proliferation of RNSCs even when EGFR/MAPK signaling is compromised (Fig. 6O and P). Collectively, these results show

We next addressed the relationship between the EGFR/MAPK pathway and the JAK/STAT pathway, which is known to play a role in RNSC self-renewal. In WT, JAK/STAT signaling is only activated in RNSCs and RBs. Interestingly, we found that JAK/STAT signaling was highly activated in clones with ectopic EGFR signaling (measured by the expression of STAT92E protein and 10  STAT92E:GFP) (Fig. 4C, D and Fig. S3). In contrast, no noticeable change of EGFR/MAPK signaling was observed in upd over-expressing clones (measured by Pnt-lacZ, data not shown). These results suggested that JAK/STAT signaling might function downstream of or in parallel to the EGFR/MAPK pathway. Although removal of STAT92E function resulted in direct differentiation of RNSCs (Singh et al., 2007), compromising STAT92E function (by a mutant or RNAi-mediated knockdown) in clones with ectopic Rafgof expression could not suppress the overproliferation phenotype, and no differentiation of RBs was observed (Fig. 5AeD). These clones resembled those with ectopic Rafgof expression. Similarly, compromising JAK/STAT activity using RNAi against Hop (the JAK kinase) and STAT92E could not suppress the over-proliferation phenotype in a Rafgof expressing background (Fig. 5E, F and Fig. S5). Thus, these genetic data suggest that the JAK/STAT pathway does not simply function downstream of EGFR/MAPK signaling. We next investigated whether EGFR signaling might act downstream of JAK/STAT signaling. While overexpression of Upd stimulates proliferation of RNSCs and promotes RB differentiation (Fig. 5G) (Singh et al., 2007), inactivation of EGFR/MAPK signaling results in proliferation arrest of RNSC (Fig. 5H). Surprisingly, when Ras function is compromised in clones ectopically expressing upd, only differentiated RC cells were observed although the ectopic RNSCs/RBs induced in the neighbourhood of upd-expressing cells were not affected, indicating that the secretion of Upd was not affected in the absence of EGFR/MAPK signaling (Fig. 5I and data not shown). These results suggest that the differentiation of RB to RC is not permanently blocked by inactivation of EGFR/ MAPK signaling. Loss of EGFR/MAPK signaling inhibits RNSC/RB proliferation, resulting in small single cells, likely RNSCs or RBs (Fig. 5J), while absence of JAK/STAT signaling leads to premature differentiation of RNSCs/RBs into RCs (Fig. 5K). Interestingly, only differentiated RCs were

EGFR/MAPK signaling acts in part through dMyc and CycE

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that dMyc and CycE mediate EGFR/MAPK signaling to regulate RNSC proliferation, which is consistent with their documented roles in the developing wing disc (Prober and Edgar, 2002). DISCUSSION Tight regulation of stem cell self-renewal versus differentiation is important to maintain tissue homeostasis. In this study, we addressed the role of EGFR/MAPK signaling in Drosophila MTs. We showed previously that EGFR/MAPK signaling functions in Drosophila ovary to restrict the germline stem cell niche activity (Liu et al., 2010). Here, we are interested in whether this signaling pathway also acts in other somatic stem cell systems, in particular the adult Drosophila MTs. We find that EGFR/MAPK signaling is essential for RNSC proliferation and RB differentiation, which is partially mediated by dMyc and CycE. EGFR/MAPK signaling in RNSC proliferation and RB differentiation Unlike ISCs in the midgut which divide daily (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006), most RNSCs exhibiting EGFR signal activation are relatively quiescent (Fig. 1C). Renal cells with compromised EGFR signaling still express the stem cell markers (e.g., STAT92E and Dl), indicating that EGFR signaling is not required for RNSC maintenance. However, ectopic EGFR signaling in these quiescent cells can drive over-proliferation of renal cells and cause tumor formation. Several lines of evidence favor the possibility that accelerated proliferation of RNSCs but not RBs induced by ectopic EGFR signaling is responsible for the overgrowth phenotypes observed, although it is possible that ectopic EGFR signaling may also force RBs to re-enter mitosis. First, under normal conditions, only RNSCs with high levels of JAK/STAT signaling proliferate. Consistently, all observed PH3-positive cells in the clones with ectopic EGFR signaling also exhibited higher levels of JAK/STAT signaling (Fig. S3). Second, we show that RNSCs, like ISCs in the midgut, express Dl and activate Notch signaling in RBs to induce differentiation. Gbe þ Su(H)-lacZ reporter is specifically detected in RBs (Li et al., 2014). To test whether ectopic EGFR signaling in RBs can lead to over-proliferation, we generated Gbe þ Su(H)-Gal4 which is specifically expressed in RBs to induce ectopic EGFR signaling. Supporting our hypothesis that RBs do not proliferate in response to ectopic EGFR signaling, no over-proliferation of renal cells was observed in these MTs (data not shown). It was shown that the EGFR pathway regulates ISC proliferation and mediates midgut homeostasis and regeneration in the adult midgut (Buchon et al., 2010; Jiang et al., 2010; Biteau and Jasper, 2011). However, unlike in MTs where ectopic EGFR signaling disrupts RB differentiation, ectopic EGFR signaling in ISCs result in accelerated ISC proliferation without disrupting EB differentiation. In WT MTs, RNSCs undergo asymmetric self-renewal divisions to generate an RNSC

daughter and an RB daughter. As a result, a labeled WT RNSC clone contains only one RNSC (Fig. 2B). Interestingly, most clones with ectopic EGFR signaling consisted of multiple RNSCs (upd-lacZ-positive, PH3-positive and Dl-positive) and RBs (Dl-negative but Gbe þ Su(H)-lacZ-positive) but no differentiated RCs, suggesting ectopic self-renewal of RNSCs and blockage of RB differentiation. It is possible that upon ectopic EGFR signal activation, asymmetric RNSC division is somehow disrupted and under some circumstances, RNSCs can occasionally undergo symmetric divisions to generate two RNSCs instead of one RNSC and one RB. A similar switch between asymmetric and symmetric division pattern was reported for Aurora-A mutant larval neuroblasts in the Drosophila brain (Lee et al., 2006). One possible explanation is that these genetic backgrounds promote equal segregation of some yet-tobe-identified cell fate determinants as shown for Aurora-A mutant larval neuroblasts and Upd, which is only expressed in RNSCs but not RBs, is a possible candidate (Singh et al., 2007). It will be interesting to test this hypothesis in the future. EGFR/MAPK signaling and JAK/STAT signaling What is the relationship between EGFR signaling and JAK/ STAT signaling? In the midgut where both pathways are activated, it was recently shown that EGFR signaling is required for ISC proliferation induced by JAK/STAT signaling under regenerative conditions (Jiang et al., 2010) and vein expression in the visceral muscles is partially under the control of JAK/STAT signaling (Biteau and Jasper, 2011). Interestingly, our genetic interaction data indicate that in MTs, EGFR/ MAPK signaling does not function in a linear hierarchy with JAK/STAT signaling. Removal of STAT92E activity in an EGFR signaling defective background promoted differentiation, suggesting that JAK/STAT signaling may function downstream of or in parallel to EGFR/MAPK signaling (Fig. 5L). However, compromising JAK/STAT activity did not suppress the over-proliferation and differentiation arrest phenotypes seen in a Rafgof background (Fig. 5CeF). Downstream mediators of EGFR/MAPK signaling Our data suggest that the effects of Rafgof in RNSC overproliferation are at least partially mediated by dMyc and CycE, two cell cycle regulators. Our results are consistent with previous data showing that in Drosophila imaginal wing discs, Ras (the immediate upstream activator of Raf) promotes cellular growth via dMyc and CycE (Prober and Edgar, 2000). Ras functions as an important signal transducer linking extracellular mitogens to intracellular mechanisms to control diverse cellular activity such as cell fate specification and proliferation. Mutations that activate Ras have frequently been identified in many kinds of human cancer (Repasky et al., 2004). In Drosophila midgut, the EGFR/MAPK pathway mediates ISC proliferation, but is not required for ISC maintenance (Buchon et al., 2010; Jiang et al., 2010; Biteau and Jasper, 2011). Similarly, our data show that RNSCs lacking Ras/Raf activity were arrested in a single cell state, suggesting

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a defect in self-renewal or a block in cell cycle progression in the absence of EGFR/MAPK signaling. Our results indicate that EGFR/MAPK signaling plays critical roles in the regulation of proliferation in various stem cell types. Therefore, our data may provide important clues for understanding mammalian kidney repair and regeneration following injury. MATERIAL AND METHODS Fly genetics Information about strains used in this study was described in the text or in Flybase. Stocks were maintained on standard cornmeal-agar medium at 25 C. Experiments were carried out at 18 C, 22 C or 25 C. y1;w1118 flies are used as wild type control. Mutants and flies used include FRT19A-Raf11, FRT19A-Dsor1LH110, FRT82B-STAT92Ej6c8, Pnt07825 (PntlacZ ), Egfrf24, FRT40A-CycEAR95, FRT82B-Rasx7b, UAS-PntP2, UAS-RasG12A, upd-lacZ, UAS-Upd, UAS-RasN17, UAS-EgfrDN, UAS-Egfr, 10  STAT:GFP, UAS-Rafgof, UAS-lamda-Egfr, UAS-PntP1, UAS-CycE, UAS-dMyc, UAS-Dap, RafRNAi (VDRC20909), Dsor1RNAi (VDRC40026), STAT92ERNAi (VDRC43866), HopRNAi (VDRC41696), EgfrRNAi (BL-25781), RasRNAi (VDRC28129), CycERNAi (VDRC47942), dMycRNAi (VDRC2948), PntRNAi (VDRC105390), and cblRNAi (BL27500). For MARCM clone analyses, the following strains were used: FRT19A, tub-Gal80; tub-Gal4, UAS-CD8GFP; hsFLP, hsFLP, UAS-CD8GFP; tub-Gal80, FRT40A; tub-Gal4, hsFLP, UAS-CD8GFP; FRT42D, tub-Gal80; tub-Gal4, hsFLP, UAS-CD8GFP; tub-Gal4; FRT82B, tub-Gal80.

Clonal analysis For AY-Gal4-mediated (Act5C-FRT > Y > FRT-Gal4, UASGFP) ectopic expression or RNAi knockdown, adult flies with proper genotypes were heat-shocked for 1 h at 37 C for consecutive 2 days. For MARCM-mediated mutant clone generation (Lee and Luo, 1999), adult flies were heat-shocked for 1 h at 37 C for four times with 8-12 h intervals. Adult flies were reared at room-temperature and dissected at the time points indicated in the text. Histology and immunohistology Immunohistology of Drosophila MTs was performed according to standard protocols with some modifications (Li et al., 2008; Liu et al., 2010). For permeabilization of Rafgof clusters, the MTs were fixed in fixative containing 0.1% Triton X-100 (4% paraformaldehyde, 0.1 mol/L Hepes pH 7.4, 0.1% Triton X-100 in PBS) for 20 min followed by rinse and washed in 0.1% Triton X-100 in PBS. For pERK staining, the MTs were fixed in fixation buffer (8% formaldehyde, 0.05 mol/L EGTA in PBS, with protein phosphatise inhibitor added, SigmaeAldrich, USA) for 30 min, rinsed in PBS containing 0.1% Tween 20 (PBST) for 1 h

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then stored in 100% methanol at 20 C overnight. Stepwise rehydration as follows: 3 parts methanol with 1 part of PBST for 10 min, 2 parts of methanol with 1 part of PBST for 10 min, 1 part of methanol with 1 part of PBST for 10 min, then PBST for 10 min. The samples were blocked in 3% BSA in PBT for 60 min, followed by normal staining procedure. The following primary antibodies were used: mouse antiArm (N2 7A1, 1:50) (Riggleman et al., 1990) obtained from Developmental Studies Hybridoma Bank (DSHB, USA), guinea pig anti-STAT92E (1:2000, generated in our lab), rabbit anti-b-galactosidase (1:5000, Cappel, USA), rabbit antiphospho-Histone H3 (1:1000, Upstate Biotechnology, USA), rabbit anti-pERK (1:200, Cell Signaling, USA), rat anti-DER (1:100) (Jekely and Rorth, 2003), mouse monoclonal antidMyc (1:5) (Moberg et al., 2004), mouse monoclonal antiCycE (gift of Hongyan Wang, 1:20) (Richardson et al., 1995), mouse monoclonal anti-Dap (gift from Maxim Frolov, 1:5) (de Nooij et al., 2000). Alexa Fluor 555/643/488 conjugated goat anti-mouse, rabbit, guinea pig and rat secondary antibodies (Molecular Probes, USA) were used to detect the primary antibodies. Samples were mounted in Vectashield mounting medium (Vector Laboratories, USA). Images were obtained using a Zeiss Upright confocal microscope (except for Fig. 3I and J, where a Nikon Eclipse microscope was used) and further processed in Adobe Photoshop and Adobe Illustrator. ACKNOWLEDGEMENTS We are grateful to Sarah Bray, David Bilder, Steve Cohen, Maxim Frolov, Ernst Hafen, Iswar Hariharan, Helena E. Richardson, Pernille Roth, Hongyan Wang, Developmental Studies Hybridoma Bank (DSHB), Bloomington stock center, NIG-FLY stock center and VDRC stock center for stocks and reagents. We thank Liwei Wang for the generation of antiSTAT92E antibody and other technical supports. We also thank Bill Chia for critical reading and comments on this manuscript. This work was supported by grants from the National Natural Science Foundation of China (No. 31271582), Temasek Life Sciences Laboratory and Singapore Millennium Foundation, and Beijing Municipal Commission of Education (No. 010135336400).

SUPPLEMENTARY DATA Fig. S1. EGFR/MAPK signaling pathway is required for RNSC proliferation. Fig. S2. Different effects of ectopic activation of Upd and Raf on RNSC proliferation and differentiation. Fig. S3. JAK/STAT signaling is activated upon ectopic expression of Raf G-O-F. Fig. S4. Multiple RBs can be observed in Raf G-O-F expressing clones. Fig. S5. EGFR/MAPK signaling functions independently of JAK/STAT signaling. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2014.11.007.

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