Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

Developmental Biology 377 (2013) 177–187

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Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila Guonan Lin, Xi Zhang, Juan Ren, Zhimin Pang, Chenhui Wang, Na Xu, Rongwen Xi n National Institute of Biological Sciences, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China

a r t i c l e i n f o

abstract

Article history: Received 10 October 2012 Received in revised form 24 January 2013 Accepted 27 January 2013 Available online 8 February 2013

Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. Here we show that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the a-integrin subunits including aPS1 encoded by mew and aPS3 encoded by scb, and the b-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a pre-requisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). Our studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations. & 2013 Elsevier Inc. All rights reserved.

Keywords: Integrin Intestinal stem cell Niche Maintenance Proliferation Basement membrane Drosophila Tumorigenesis

Introduction Adult stem cells or tissue-specific stem cells are typically associated with a specific microenvironment or niche, which provides critical signals to direct their self-renewal and multiple lineage differentiation (Jones and Wagers, 2008; Li and Xie, 2005; Morrison and Spradling, 2008). Consequently, proper association or anchorage between the stem cell and its niches is critical for the maintenance and function of the stem cell. Emerging evidence has suggested a primary role of cell adhesion molecules in anchoring stem cells to their respective niches. Direct cell-tocell adhesion by adherens junctions has been implicated as a strategy for stem cell anchorage to the niche cells. On the other hand, integrin-mediated adhesion is also involved in positioning stem cells to the basement membrane, and diverse integrin subunits could be utilized for different types of stem cells (Jones and Wagers, 2008; Li and Xie, 2005; O’Reilly et al., 2008; Song et al., 2002; Tanentzapf et al., 2007; Xi, 2009).

n

Corresponding author. E-mail address: [email protected] (R. Xi).

0012-1606/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ydbio.2013.01.032

The adult Drosophila midgut has recently emerged as an attractive system to study the stem cell–niche interaction and the role of cell adhesion molecules (Biteau et al., 2011; Karpowicz and Perrimon, 2010; Sahai-Hernandez et al., 2012). The midgut epithelium is single-layered with a simple stem cell lineage (Fig. 1A) (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). The epithelium is surrounded by the visceral muscle cells and separated from them by a thin layer of basement membrane that composed of extracellular matrix (ECM). The intestinal stem cell (ISC) located basally to the basement membrane and marked by the expression of the Notch (N) ligand Delta (Dl), is the only epithelial cell that is capable of cell division. Typically after each cell division, each ISC renews itself and produces another daughter named enteroblasts (EB), which is committed to differentiate into either an absorptive enterocyte (EC) or a secretory enteroendocrine (ee) cell. Previous studies suggest that the visceral musculature forms a regulatory niche for ISCs by producing several signal molecules, including Wingless (Lin et al., 2008), Unpaired (Lin et al., 2010), and Vein (Biteau and Jasper, 2011; Buchon et al., 2010; Jiang et al., 2011; Xu et al., 2011) that, respectively activates Wnt, JAK/STAT and EGFR signaling pathway to promote maintenance and proliferation of ISCs, and insulin-like peptide Ilp-3 in response to feeding to coordinate ISC activity with nutrition status (O’Brien

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Fig. 1. Expression pattern of integrins in the midgut. (A) A diagram showing a cross-section of the Drosophila midgut epithelium. Abbreviations: BM, basal membrane; ISC, intestinal stem cell; EB, enteroblast; EC, enterocyte; and ee, enteroendocrine cell. (B) RT-PCR analysis of transcripts of all integrin subunits in the midgut. (C)–(E) Crosssections stained with anti-Mew (aPS1, (C) and (D), red) and anti-Scb (aPS3, (E), red). esg4 GFP (green) marks ISCs and EBs, Dl-lacZ marks ISCs, and F-actin marks the muscle fiber. (C0 ), (D0 ) and (E0 ) red/ green channels of (C), (D) and (E), respectively. (F) and (G) A cross-section (F) and a superficial section (G) through the epithelium layer stained with anti-Mys (bPS integrin, red) showing that Mys was accumulated at the basement membrane and the membrane of ISCs and EBs. (F0 ) The red channel of (F). In all images, DAPI staining was in blue. Scale bar, 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

et al., 2011). Therefore, basal localization could be important for ISCs to properly receive these signals for their maintenance and function. Integrin-mediated cell adhesion is commonly involved in cell attachment to the ECM. Integrins are heterdimer receptors consisting of one a subunit and one b subunit. The complex acts as a docking site to link the extracellular ECM molecules to the intracellular cytoskeleton. It also interacts with growth factor receptors and other effectors to regulate cell survival, proliferation and differentiation (Hynes, 2002). In Drosophila, there are five a integrin subunits: aPS1 (encoded by the gene multiple edematous wings, mew), aPS2 (encoded by inflated, if), aPS3 (encoded by scab, scb), aPS4 and aPS5 as well as two b integrin

subunits: bPS (encoded by the gene myospheroid, mys) and bn (Bokel and Brown, 2002). In the Drosophila ovary, integrin-mediated cell adhesion is required for follicle stem cell maintenance in the germarium. Specifically, a1b integrins and a2b integrins are important for both positioning and proliferation of follicle stem cells (O’Reilly et al., 2008). In the Drosophila testis, b integrin and Lasp, a novel regulator of integrin, are required for anchoring the germline stem cell niche (or hub) to the anterior tip, and integrin dysfunction leads to hub detachment and consequently stem cell delocalization (Lee et al., 2008; Tanentzapf et al., 2007). Notably, although bPS is widely expressed in Drosophila tissues and required in many developmental processes, bn expression is more restricted, such that it is highly expressed in gut tissues during developmental stage

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as well as in adulthood. bn mutant flies are viable and display no obvious phenotype, except that mutant together with bPS cause defects in midgut migration (Devenport and Brown, 2004), suggesting that bn contributes to midgut migration during embryogenesis. However, whether bn functions in adult intestine has not yet been studied. In this paper, we have analyzed the functions of each integrin subunits and downstream regulators in regulating ISCs in the midgut. We found that aPS1, aPS3 and bPS integrin subunits are critical for both maintenance and proliferation of ISCs, while bn only contributes to ISC maintenance when bPS is disrupted. In addition, integrin-mediated adhesion is also important for epithelial proliferation induced by various mitogenic signals, indicating a critical role of integrin in mediating epithelial regeneration and tumorigenesis.

Results Expression of integrins in the midgut We first examined the expression of integrin subunits in the midgut by RT-PCR analysis. Transcripts of all known a subunits, except a5, and b subunits were detected (Fig. 1B). We further examined the localization of two a integrin subunits by staining with specific antibodies. The binary system esgGAL4, UAS-GFP (esg4GFP) was used to mark ISCs and EBs with GFP (Brand and Perrimon, 1993). The a1 integrin, Mew, was strongly accumulated on the basal membrane of esg 4GFP þ cells contacting ECM, but was barely detected in any other regions along the interface between the muscle cells and the differentiated epithelial cells (Fig. 1C). Co-staining with Dl-lacZ, which specifically marks ISCs, confirmed that Mew was strongly enriched on the basal membrane of ISCs facing the muscle layer (Fig. 1D). The a3 integrin, Scb, in contrast, was generally enriched along the interface between the muscle layer and the epithelium (Fig. 1E). Immunostaining with antibodies against bPS integrin Mys showed that Mys was strongly

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accumulated at the basement membrane and specifically at the membrane of ISCs and EBs, but weakly in ECs (Fig. 1F and G). The expression analysis indicates that multiple integrin subunits are richly expressed in ISCs and may be involved in the regulation of ISC.

a1, a3 and bPS integrins are required for ISC maintenance Integrins, except bn, are essential for viability, and the homozygous mutants cannot survive to adulthood. To examine the cellautonomous requirement of integrin in ISC maintenance, we studied ISC behavior in GFP-labeled, homozygous mutant ISC clones induced by the MARCM system (Lee and Luo, 2001), as previously described (Lin et al., 2008, 2010; Xu et al., 2011). Briefly, wild type or mutant ISCs with GFP expression can be randomly generated by induced-mitotic recombination. Normally, each ISC divides approximately once a day, and the GFP marked ISC will give rise to a cluster (or a clone) of GFP positive cells that contains the originally marked ISC as well as the differentiated cells derived from it. Therefore, examining the maintenance of ISCcontaining clones (or ISC clones hereafter, for simplicity) at different time points after clone induction (ACI) would be reflective of ISC maintenance status during this period. In wildtype control, at two weeks ACI, the GFP-marked ISC clones were found to be scattered along the midgut epithelium. Within each clone, the Dl þ ISC and the differentiated cells, including EC, recognized by their large polyploid nuclei, and ee cell, marked by Prospero (Pros) expression, could be observed (Fig. 2A and B and data not shown). For wildtype ISC clones, approximately 94.8% observed on day 4 ACI were maintained on day 14 ACI (Fig. 2A and Table 1), which is consistent with previous observations that normal ISCs have a slow turn-over rate. mysXG43 and mysM2 are strong loss-of-function alleles of mys (Bunch et al., 1992; Jannuzi et al., 2002; Levi et al., 2006). mys mutant ISC showed a rapid ISC clone loss phenotype (Fig. 2C), with only about 26.9% and 42.7% ISC clones maintained 2 weeks ACI, respectively

Fig. 2. Integrins are essential for ISC maintenance. (A)–(L) GFP clones (green) of given genotypes in the posterior midgut at two weeks after clone induction (ACI). Most of mys, mew, and scb mutant clones were no longer maintained at day 14 ACI. (B), (D), (F), (H), (J), (L) Typical GFP clones for (A), (C), (E), (G), (I) and (K), respectively. In all images, anti-Dl staining was in red and DAPI staining was in blue. Scale bars in (A), 100 mm, and in (B), 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Integrin-mediated cell adhesion is required for ISC maintenance. Day 4

Day 7

Day 14 Normalized maintenance rate (%)

Genotype

Average no. ISC clone per gutn

No. Gut (n)

Normalized maintenance ratenn (%)

Average No. ISC clone per gut

No. Gut (n)

Normalized maintenance rate (%)

Average No. ISC clone per gut

No. Gut (n)

wt (FRT19A) wt (FRT79D) mysM2 FRT19A mysXG43 FRT19A mysM2 FRT19A; UAS-mys mysXG43 FRT19A; UAS-mys mewM6 FRT19A mewP13 FRT19A ifk27e FRT19A ifB2 FRT19A mewM6ifk27e FRT19A mewM6ifk27e FRT19A; UASmew FRT42B scb1 FRT42D scb2 talin1 FRT79D talin2 FRT79D talin79 FRT79D ilk1 FRT79D lanA12641 FRT79D lanA9-32 FRT79D

13.7 7 1.49 21 7 1.68 6.2 7 0.74 19.3 7 1.48 10.9 7 0.90

14 10 12 10 10

100.07 10.85 100.07 8.00 100.07 11.94 100.07 7.65 100.0 7 8.26

13.7 72.09 21.1 71.46 4.7 70.70 8.7 71.48 10.5 70.89

14 10 10 10 10

100.07 15.22 100.57 6.97 76.2 7 11.35 45.1 7 6.13 96.3 7 8.12

13.0 7 1.84 19.7 7 1.59 2.6 7 0.43 5.2 7 1.19 10.8 7 0.98

14 10 11 10 10

94.87 13.44 93.87 7.58 42.77 7.00 26.97 6.17 99.17 8.94

16.6 7 1.75

10

100.07 10.52

15.8 71.46

10

95.2 7 8.79

13.5 7 0.89

10

81.37 5.33

11.8 7 1.51 7.6 7 0.91 14.4 7 1.84 7.8 7 1.07 15.6 7 2.35

13 11 13 10 10

100.07 12.78 100.07 12.75 100.07 12.81 100.07 13.76 100.07 15.08

7.1 70.62 3.8 70.59 15.1 71.86 7.6 71.26 10.5 71.19

15 10 14 11 10

60.27 5.20 50.77 7.90 104.8 7 12.97 97.9 7 16.15 67.3 7 7.66

3.7 7 0.55 1.9 7 0.25 13.8 7 1.46 7.1 7 0.97 7.4 7 1.10

15 10 12 10 11

31.57 4.62 25.27 3.26 95.67 10.13 91.07 12.45 47.27 7.04

13.6 7 1.23

12

100.07 9.08

11.9 71.23

13

83.4 7 8.71

11.2 7 0.83

12

82.27 6.14

23.6 7 1.90 21.3 7 2.44 5.4 7 0.87 6.8 7 0.66 3.2 7 0.70 17.3 7 1.21 17.3 7 2.16 17.9 7 2.86

13 11 10 12 10 10 13 14

100.07 8.04 100.07 13.16 100.07 16.14 100.07 9.68 100.07 21.75 100.07 7.00 100.07 12.49 100.07 16.00

19.9 71.84 17.2 71.64 2.73 70.60 4.3 70.69 0.7 70.21 3.6 70.43 12.2 71.76 15.3 72.60

13 12 11 15 10 10 10 13

84.4 7 7.81 71.5 7 6.82 50.57 11.19 63.5 7 10.17 21.9 7 6.67 20.87 2.47 70.57 10.15 85.7 7 14.55

15.8 7 1.70 6.7 7 0.93 1.18 7 0.26 1.7 7 0.30 0.3 7 0.14 1.5 7 0.58 10.9 7 1.92 10.2 7 1.61

14 11 11 10 11 10 10 13

66.83 7 7.21 28.07 3.86 21.97 4.88 24.97 4.39 8.57 4.40 8.77 3.36 62.97 11.10 56.97 9.01

An ISC clone is defined as a GFP clone that contains at least one Dl þ ISC. The normalized maintenance rate at a given time point is calculated as the number of ISC clones per midgut divided by the average number of ISC clones per midgut on day 4. Mean 7SEM were shown. n

nn

(Table 1). Many GFP clones remained on day 14 showed smaller size phenotype and contained only differentiated cells, suggesting that the previous ISC in the clone was recently lost (Fig. 2D). To further exclude the possibility that the ISC loss is caused by a background mutation, we constructed and expressed a UAS-mys transgene with full length mys cDNA. This transgene expression efficiently rescued the ISC loss phenotype (Fig. 2E and F and Table 1). We therefore conclude that mys is required for ISC maintenance. Since a1-4 integrins are all expressed in the midgut, we attempted to examine the function for each one of them. There is no mutant allele for a4 available, which prevented our analysis for this subunit. MARCM clonal analysis for the rest three subunits were conducted using strong or null alleles for each subunit. ISC clones homozygous for mewM6 and mewP13 (Brower et al., 1995; Levi et al., 2006; Prokop et al., 1998) showed significant loss phenotype (Fig. 2G and H), with only 31.5% and 25.2% ISC clones remained on day 14 ACI, respectively. ISC clones carrying scb1 or scb2 (Araujo et al., 2003; Stark et al., 1997) had a similar, although weaker, clone loss phenotype (Fig. 2K and L), with about 66.8% and 28.0% ISC clones remained on day 14 ACI, respectively. In contrast, mutation in a2 (if) did not show any obvious maintenance defect (Fig. 2I and J), with more than 90% ISC clones still remained on day 14 ACI for both ifk27e and ifB2 (Brower et al., 1995; Levi et al., 2006) homozygous clones (Table 1). These observations suggest that a1 and a3 PS integrins are required for ISC maintenance whereas a2 is dispensable. Because a1 and a2 integrin subunits are both required for maintaining follicle stem cells in the ovary, we tested whether a stronger ISC loss phenotype could be observed when mew and if are simultaneously mutated. The mew if double mutant ISC clones showed a similar ISC loss rate to mew single mutant ISC clones (Table 1), and expression of a UAS-mew transgene could efficiently rescue

the ISC loss phenotype. These data strongly support the notion that mew but not if is required for ISC maintenance in the Drosophila midgut. Taken together, these data demonstrate that a1b and a3b integrins are the major integrins required for ISC maintenance. Function of bn PS integrin in ISC maintenance Because bn integrin is specifically enriched in the midgut, it might have a significant role in ISCs. As previously reported, homozygous bn mutants are viable (Devenport and Brown, 2004). Interestingly, adult midguts from the mutants are morphologically indistinguishable to the wildtype ones. In addition, the proportion of ISCs in the intestinal epithelium is also largely normal, as the percentage of Dl þ ISCs was approximately 9.9% in bn homozygous intestines, compared to 10.7% in the control heterozygous intestines (Supplemental Fig. S1A–C). Previous studies showed that in the developing midgut, mys can fully compensate for the integrin function when bn is lost, and the requirement of bn can be reflected when mys is simultaneously disrupted (Devenport and Brown, 2004). To test whether they have a redundant role in the adult intestine, we removed one functional copy of mys in bn homozygous mutant flies. In mys-/þ; bn/bn mutant intestines, the percentage of Dl þ ISCs is approximately 10.6%, similar to the percentage in the control intestines (Supplemental Fig. S1C). To further test its potential function, we knocked down bn in the mys homozygous mutant ISCs by expressing UAS-bn-RNAi, and tested whether reduced function of bn could enhance the ISC loss phenotype caused by mys mutation. As a control, ISC clones with bn-RNAi had no obvious effect on ISC maintenance. However, a slightly stronger ISC loss phenotype was observed for ISC clones with both mys mutation and bn-RNAi

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(Supplemental Fig. S1D–F). These data indicate that bn is normally dispensable for ISC maintenance, yet may contribute to integrin function for ISC maintenance when Mys is not functional. To further address the functional relationships between bn and mys, we overexpressed bn in mys mutant clones. Overexpression of bn partially rescued the ISC loss phenotype (Supplemental Fig. S1F), suggesting that bn has certain overlapping function with mys but cannot fully replace mys for ISC maintenance.

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Integrin-mediated cell adhesion is required for ISC maintenance To further test whether integrin-mediated adhesion is required for ISC maintenance, we examined the function of intracellular ligand and intercellular integrin signaling components. Laminin A (lanA) is known as the primary ligand for integrin (Henchcliffe et al., 1993). Mutation of lanA resulted in mild ISC loss (Fig. 3C), with about 62.9% and 56.9% ISC clones maintained, respectively for

Fig. 3. Integrin mediated-adhesion is essential for ISC maintenance, but not survival. (A)–(H) GFP clones of given genotypes in posterior midgut at two weeks ACI. (B), (D), (F) and (H) Typical GFP clones for (A), (C), (E) and (G), respectively. I, Experimental control of esgGal4, UAS-GFP, Gal80ts at 29 1C for 2 weeks. (J)–(K), In mys and talin knockdown guts at 29 1C for 2 weeks, many esg 4GFP þ cells were lost. (L) A graph showing the percentage of esg 4GFP þ cells in the midgut of given genotype. Mean7 SEM were shown. n¼10 sections. Values significantly different in a student’s t-test (***P o 0.001). (M)–(O), TUNEL assay (red) in midguts of given genotypes. Apoptotic cells was occasionally observed in GFP negative cells in wildtype control ((M), esgGal4 ts, UAS-GFP) or mys-RNAi midgut ((N), arrowhead). Apoptotic cells in GFPþ population were significantly increased after UAS-rpr-mts expression ((O), arrowheads, note that GFP expression was reduced in the dying cells upon rpr induction). In all images, DAPI staining was in blue. Scale bar in (A), 100 mm, and in (B) and (I), 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Integrins are required for ISC proliferation. (A) Quantification analysis of cell number of ISC clones at day 14 ACI. n ¼10–30 clones. (B) Quantification analysis of pH3 þ cells in Dl þ cells in wildtype and mys knockdown midguts. n¼10 sections. Statistics by a student’s t-test (**P o0.01, ***P o0.001). (C)–(E) FACS analysis of dissociated guts. In w1118 fly midgut (C), all cells did not show any GFP signal in boxed area. In experimental control (D) and mys knockdown guts (E), the GFP-positive cells in boxed area were used for DNA dye profiling. (D0 ) and (E0 ), Cell cycle profiling of (D), (E) boxed cells, respectively. Red lines are computer model for G1-phage cells, green lines for S-phase cells, and blue lines for G2/M-phase cells. (F) N knockdown clone on day 14 ACI developed ISC-like tumors. (G) N knockdown in mys mutant clones largely prevented tumor development. (H) a tumor from N knockdown clone. (I) a tumor from N mys double mutant clone. Anti-Dl and anti-Pros staining was in red and DAPI staining was in blue. Scale bar in (F) 100 mm, and in (H) 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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lanA12641 and lanA9–32 mutant clones at two weeks ACI, compared to 93.8% for the wildtype control (Table 1). Enriched LanA protein was still detected, although reduced in the basal membrane that was associated with lanA mutant clones (supplemental Fig. S3), suggesting that LanA is produced by ISCs as well as other cells. One likely candidate is the underlying muscle cells. The redundant source of LanA could explain the relative weak maintenance phenotype caused by lanA mutation. Talin/rhea and integrin-linked kinase (ilk) are integrin binding linkers, which function downstream of integrin to connect integrin to actin cytoskeleton (Brown et al., 2002; Dedhar et al., 1999). Their functions are essential for integrin-mediated adhesion in Drosophila, but usually are not required for cell differentiation (Brown et al., 2002; Zervas et al., 2001). The loss of talin or ilk caused a rapid ISC loss phenotype (Fig. 3E–H), with only 21.9%, 24.9%, 8.5% and 8.7% ISC clones maintained, respectively for talin1, talin2, talin79 and ilk1 mutant clones (Table 1). Moreover, by using esgGAL4 to drive UAS-mysRNAi or UAS-talin-RNAi in ISCs and EBs, rapid ISC loss was also observed (Fig. 3I–K). Normally, approximately 27.2% of total epithelial cells are esg4GFP þ cells, which are uniformly scattered in the epithelium (Fig. 3I). However, esg4GFP þ cells and Dl þ cells were dramatically decreased in mys or talin RNAi intestines in two weeks after shifting to the restrictive temperature, with only 1.4% and 9.5% esg4GFP þ cells remained, respectively for two independent mys-RNAi lines, and 9.3% and 2.0%, respectively for two independent talin-RNAi lines (Fig. 3L). We therefore conclude that ISC-ECM adhesion mediated by integrin signaling is essential for ISC maintenance. Integrin signaling is dispensable for multiple lineage differentiation In addition to cell attachment, integrin mediated cell adhesion has also been implicated in cell survival, cell proliferation and differentiation. Detachment from ECM sometimes induces apoptosis termed anoikis. The time-course clonal analysis of integrin mutant clones showed that the recently lost ISCs seem to generate a small clone of differentiated cells, suggesting that the lost ISCs are not eliminated by cell death but rather have initiated differentiation (Figs. 2 and 3). TUNEL labeling experiment to detect apoptotic cells revealed that apoptotic cells were rare in normal intestinal epithelium (Fig. 3M) and in mys-RNAi treated epithelium (Fig. 3N), but were readily observed when the cell death inducer reaper (rpr) was expressed (Fig. 3O). Furthermore, integrin mutant ISCs did not show any obvious sign of apoptosis (0/52 for mysXG43, 0/39 for mysM2, 0/51 for mewM6, 0/37 for mewP13, 0/107 for scb1 and 0/74 for scb2) (data not shown), suggesting that integrin is not required for ISC survival. Consistent with this notion, mys-RNAi-induced ISC loss could not be rescued by forced expression of P35 (Fig. 3L), a potent baculovirus caspase inhibitor of apoptosis (Clem et al., 1991; Hay et al., 1994). Normally, when a new EB is produced after a recent ISC division, at about 90% of chance, it will differentiate into an EC, and 10% of chance it will adopt an ee cell fate (Ohlstein and Spradling, 2007). To test if integrin is required for multiple lineage differentiation and the binary fate choice, we analyzed the proportion of ee cells to ECs in integrin mutant ISC clones. The ee cells can be specifically marked by Prospero (Pros), a transcription factor localized in the nucleus (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006), and ECs are characterized by the large polyploid nuclei. In both wild type and integrin mutant ISC clones, ee cells and ECs were readily observed, many ee cells could also express a terminal differentiation marker, Tachykinin (Dtk) (Ohlstein and Spradling, 2006) (Supplemental Fig. S2B), suggesting that integrin is not required for multiple lineage differentiation. Furthermore, the proportion of ee to EC population in integrin mutant ISC clones was about 10.8%, 11.8%, 12.2%, 10.0% and 9.0%,

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respectively for mysXG43, mysM2, mewM6, mewP13 and scb2 mutant clones, similar to that in wildtype ISC clones (10.8%) (Supplemental Fig. S2C), suggesting that the binary fate choice of EB during lineage differentiation is not affected by the disruption of integrin function. One exception is ilk, depletion of which caused arrest or delay of cell differentiation, as the cells in the clones were either diploid cells or immature ECs (Fig. 3G and H). But this probably suggests an integrin-independent function of ilk that is important for ISC differentiation. Hence, we conclude that integrin signaling is dispensable for multiple lineage differentiation from progenitor cells. Integrin signaling is required for ISC proliferation Along with the maintenance phenotype, we also noticed that integrin mutant ISC clones were generally smaller than the wild type ISC clones as there were fewer cells within each clones (Figs. 2 and 3). The wild type, two-week-old ISC clones contained approximately 21.1 cells on average (Fig. 4A), whereas integrinsignaling mutant ISC-containing clones that remained at two weeks ACI only contained less than a half (Fig. 4A). The smaller clone size could be caused by reduced proliferation rate of integrinmutant ISCs. We found that there was a significant decline of ISCs with positive phosphor-histone3 (PH3) staining in esgGAL4, UASmys-RNAi midguts compared to wildtype ones (Fig. 4B), suggesting that integrin signaling is required for ISC proliferation. To determine if there is a specific defect in cell cycle progression of integrin mutant ISCs, we flow the sorted esg 4GFP þ cell in esgGAL4, UAS-mys-RNAi midguts, followed by DNA profiling. Similar to a previous observation (Amcheslavsky et al., 2011), the majority of wild type esg 4GFP þ cells were in G1 phase, with a small percentage in S and G2 phase (Fig. 4D). The cell cycle profile of esg4GFP þ cells with mys-RNAi is largely similar, except that significantly more cells are delayed or arrested in S phase (Fig. 4E), suggesting that integrin signaling is required for S phase progression during ISC division. To further test the requirement of integrin signaling in ISC proliferation and maintenance, we asked whether integrin is required for ISC-like tumor development caused by the loss of Notch (N). N is essential for lineage differentiation and disruption of N in ISCs results in accumulation of ISC-like cells, which eventually develop into tumor-like masses (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Using the MARCM system, we expressed N-RNAi in randomly-induced, GFP-marked clones, and each ISC clone was able to develop into an ISC-like tumor (Fig. 4F), as previously described (Xu et al., 2011). When N-RNAi was expressed in GFP-marked mys mutant clones, many clones were eliminated from the epithelium on day 14 ACI (Fig. 4G). The ISC-like tumors still could be observed, although rarely, but the size of the tumors was significantly smaller (Fig. 4H and I), suggesting that mys is required for both maintenance and propagation of N-mutant tumors. Taken together, those data further support the hypothesis that integrin signaling is required for both maintenance and proliferation of ISCs in the Drosophila midgut. Integrin signaling has a permissive role for ISC proliferation induced by local proliferative signals Previous studies have shown that ISC proliferation in the Drosophila midgut can be regulated by Wg and JAK/STAT and EGFR signaling pathway activities (Beebe et al., 2010; Biteau and Jasper, 2011; Buchon et al., 2010; Jiang et al., 2011; Lee et al., 2009; Lin et al., 2008, 2010; Xu et al., 2011). We therefore tested whether forced activation of these signaling pathways could promote ISC proliferation when integrin is depleted. We generated integrin mutant ISC clones co-expressing an active form of EGFR or Ras.

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Fig. 5. ISC proliferation induced by local signals requires integrin. (A)–(C) GFP clones of given genotypes in posterior midgut at two weeks ACI. Note that over-activated EGFR, JAK/STAT or Wg signaling in integrin mutant clones could not rescue ISC proliferation defect. (D) Quantification analysis of cell number of given genotype ISC clones on day 14 ACI. Mean7 SEM were shown. n¼ 10–30 clones. Values significantly different in a student’s t-test (***Po 0.001). (E)–(F) Superficial sections through the epithelium from esgGal4, UAS-GFP, UAS-mys-RNAi/ þ (E) and esgGal4, UAS-GFP, UAS-mys-RNAi/þ , UAS-Ras/þ (F) flies at 29 1C for two weeks. Note that Ras overexpression can not rescue ISC loss caused by mys knockdown. In all images, DAPI staining was in blue. Scale bar in (A) 100 mm; and in (E) 10 mm.

Interestingly, activation of EGFR signaling could not rescue the proliferation phenotype. The size of ISC clones was also similar to the clones without EGFR or Ras activation. In addition, the ISC clones were gradually lost at a similar rate to mys ISC clones (Fig. 5A and D). Consistent with this observation, ISC loss in esgGAL4, UAS-mys-RNAi midguts could not be rescued by co-expression of UAS-RasV12 (Fig. 5E and F). Similarly, expression of an active form of Arm, or the JAK/STAT signaling ligand Upd in mys mutant ISC clones was unable to rescue the proliferation phenotype (Fig. 5B–D). These data suggest that integrin has a permissive role for ISC proliferation in response to local proliferative signals. Because integrin mutant ISCs could not proliferate even under hyperactivation of several proliferation-promoting signaling pathways, we further asked whether integrin is also required for intestinal hyperplasia induced by the loss of adenomatous polyposis coli (Apc) (Lee et al., 2009). Genetic studies have suggested that

Wnt signaling hyperactivation, which drives ISC proliferation, is primarily responsible for the hyperplasia, although additional mechanisms could be involved (Lee et al., 2009). Apc Apc2 double mutant clones displayed a strong over-proliferation phenotype, as the clone size increased dramatically over time, and eventually the epithelia became multi-layered (Fig. 6B), as previously observed (Lee et al., 2009). Strikingly, co-expressing mys-RNAi in Apc Apc2 double mutant clones strongly inhibited the clonal expansion and multi-layer formation (Fig. 6C), showing that integrin is necessary for the development of intestinal hyperplasia after the loss of Apc.

Discussion This study demonstrates a requirement for integrin in maintaining midgut ISCs in Drosophila. Several integrin subunits and integrin signaling components are expressed at high levels in ISCs

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Fig. 6. Integrin is required for the development of intestinal hyperplasia after APC loss. (A)–(C) GFP clones of given genotypes in posterior midgut at two weeks ACI. (A0 )—(C0 ) Cross sections of typical GFP clones for (A)–(C), respectively. In all images, DAPI staining was in blue. Scale bar in (A) 100 mm; and in (A0 ) 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

but low levels in differentiated cells, suggesting that stem cells themselves actively produce integrin and ECM components to assemble integrin-mediated adhesion to ECM. We suggest that this mechanism enables stem cells to be tightly associated with the local niche, which is essential for their long-term maintenance and function. In the Drosophila ovary, follicle stem cells also autonomously produce laminin A for integrin-ECM establishment to fix their position at the right place (O’Reilly et al., 2008), suggesting that this mechanism is conserved in Drosophila epithelial stem cells. Enrichment of specific integrin subunits was frequently observed in many mammalian adult stem cells (Watt, 2002). Although genetic evidence supporting their functional significance in mammals is limited due to functional redundancy of integrin species, there are a few examples, such as in the mammary gland, disrupting b integrin function at the basal compartment is sufficient to disrupt mammary stem cell (Taddei et al., 2008). Therefore, integrin-mediated cell adhesion could be an evolutionarily conserved mechanism for stem cell anchorage to the niche microenvironment by establishing stem cell-ECM adhesion. As the intestinal epithelium is constantly under physical compression or stretch enforced by the surrounding muscles, it is imaginable that ISC could solicit additional means for keeping ISCs within the niche. In the Drosophila ovary, follicle stem cells are also in contact with escort cells (Nystul and Spradling, 2007) in addition to the basement membrane. Moreover, DE-cadherinmediated adhesion between follicle stem cells and escort cells are important for follicle stem cell maintenance (Song and Xie, 2002). These studies suggest that in addition to the function of integrinmediated adhesion to ECM in positioning follicle stem cells, DE-cadherin-mediated adhesion between FSC and escort cell may also contribute to positioning follicle stem cells. Interestingly, in the midgut, DE-cadherin has a specific enrichment

between ISCs and neighboring non-stem cells, including EBs and enterocytes (Maeda et al., 2008). It would be interesting to study whether DE-cadherin-mediated adhesion between ISC and nonstem cells also contributes ISC maintenance in the midgut. Our study also reveals an essential role of integrin in promoting ISC division and in mediating intestinal tumorigenesis. This requirement for cell proliferation was also observed for ovarian FSCs, suggesting a general role of integrin in stem cell proliferation in diverse epithelial tissues. However, the underlying mechanism is still poorly understood. As we have shown, forced activation of EGFR, Wg or JAK/STAT signaling, or loss of Apc, all of which are known to potently induce ISC proliferation and epithelial hyperplasia, are unable to do so if integrin is disrupted. We propose that disruption of adhesion to ECM could trigger an ‘‘adhesion checkpoint’’ for ISC division. Therefore, integrin could be important for stem cells to sense the environment to allow cell cycle progression. Alternatively, integrin may play a key role in orientating mitotic spindle to determine symmetric or asymmetric cell division, as integrin signaling can control cell division axis during cell mitosis (Toyoshima and Nishida, 2007). However, although after each typical ISC division, one of the daughters adopts ISC fate while the other adopts EB fate and then commits differentiation, there is no obvious correlation between the orientation of mitotic spindle and symmetric or asymmetric ISC divisions (Ohlstein and Spradling, 2007). Therefore, the role of mitotic spindle orientation on stem cell self-renewal remains to be determined. Goulas et al. (2012) recently showed that disruption of integrin expands ISC population because of increased symmetric divisions The reason of these seemingly contradictory results from their and our studies is unclear, but there are several possibilities, including influences of different mutant alleles, different genetic background, or potential off target effects by

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RNAi. In addition, the RNAi approach, which is primarily used in their studies, might facilitate the discovery of its role in asymmetric cell division, as unlike genetic null alleles, it may allow residual integrin function sufficient for ISC maintenance without affecting ISC proliferation. In summary, this study reveals novel roles of integrin-mediated cell adhesion in ISC maintenance and proliferation in the Drosophila midgut. Our studies suggest that integrin disruption may trigger an adhesion checkpoint for cell cycle progression of ISCs, and therefore integrin could be an indispensable player during intestinal tumorigenesis. The pleiotropic function of integrin indicates that it could serve as a functional platform to coordinate ISC maintenance, proliferation and differentiation in response to environmental cues. Given evolutionary conservation of integrin function, it could have a similar role in epithelial stem cells of mammals as well. As integrin has been considered as a potential pharmaceutical target because of its essential role in angiogenesis (Waldner and Neurath, 2010), it would be interesting to investigate whether targeting integrin could also trigger ‘‘adhesion checkpoint’’ in various tumors, such as gastrointestinal tumors, for cure.

Materials and methods Fly stocks The following fly stocks were used: Mutant alleles on FRT: mewM6FRT19A; mewP13 FRT19A; ifk27e FRT19A; mewM6 ifk27e FRT19A; ifB2 FRT19A; FRT42B scb1; FRT42D scb2; mysM2 FRT 19A (gifts from Mark Krasnow and Alana O’Reilly) (Levi et al., 2006; O’Reilly et al., 2008); mysXG43 FRT 19A; talin1 FRT2A; talin2 FRT2A (gift from Jean-Rene´ Huynh); talin79 FRT2A (gift from Mark Krasnow); ilk1 FRT2A; lanA12641 FRT2A; lanA9-32 FRT2A (gifts from Alana O’Reilly) (O’Reilly et al., 2008); bn1 (gift from Jean-Rene´ Huynh); and FRT82B Apc2g10ApcQ8 (gift from M. Piefer). MARCM stocks: hsflp122, tub-gal80 FRT19A/ FM7; Act-Gal4, UAS-GFP/ Cyo; hsflp122, tub-Gal4, UAS-GFP/ FM7; tub-gal80 FRT42B/ Cyo; hsflp122, tub-Gal4, UAS-GFP/ FM7; tub-gal80 FRT42D/ Cyo; hsflp122, tub-Gal4, UAS-GFPnls/ FM7; tub-gal80 FRT2A/ TM3; and hsflp122, tub-Gal4, UAS-GFPnls/ FM7; FRT82B tub-gal80/ TM3. RNAi transgenes: UASmys-RNAi (Vienna Drosophila RNAi center, VDRC# 29613); UASmys-RNAi (Bloomington stock center, BSC, TRiP line# 27735); UAS-talin-RNAi (VDRC# 40399); UAS-talin-RNAi (BSC, TRiP line# 28950); UAS-bn-RNAi (VDRC# 893); and UAS-N-RNAi (NIG-Fly, # 3936-R2). UAS transgenes: UAS-bn; UAS-P35; UAS-rpr-mts (Sandu et al., 2010) (gift from H. Steller); UAS-armDN (gift from A. Martinaz-Arias); UAS-upd(PK9) (gift from D. Harrison); UASRas85Dv12 (Lee et al., 1996); UAS-EGFRltop (Queenan et al., 1997), and UAS-mew (gift from Akira Chiba). The construct of UAS-mys was made by inserting the full length of wild-type mys cDNA to the pUAST plasmid and the P-element transformation was obtained by standard methods. Reporters and Gal4 lines: esgGal4,UAS-GFP (gift from S. Hayashi). Flies were maintained at 25 1C on standard media, unless otherwise stated.

to 29 1C for one day, and the posterior 1/3 region of posterior midgut was analyzed. The other part of the midgut shows severe ISC loss phenotype, which is presumably caused by leaky expression of rpr at early developmental stages. All of the graphs were made using windows Prism 4 software. Immunofluorescence The intestines were dissected in Insect Medium. Gut fixation and immunostraining were as previously described (Lin et al., 2008), except for anti-Mew antibody staining, in which case samples were fixed in 4% formaldehyde for 30 min. Following antisera and dyes were used: mouse anti-Dl antibody (Developmental Studies Hybridoma Bank (DSHB); 1:100); mouse anti-Pros antibody (DSHB, 1:300); mouse anti-Mew antibody (DSHB, 1:300); rabbit anti-Scb antibody (a gift from Shigeo Hayashi, 1:1000); mouse anti-Mys antibody (DSHB, 1:300); rabbit anti-LanA antibody (a gift from Stefan Baumgartner, 1:1000); rabbit anti-Tachykinin antibody (a gift from Dick Na€ssel, 1:3000); rabbit anti-phospho-Histone H3 antibody (Upstate, 1:1000); rabbit polyclonal anti-b-gal antibody (Cappel, 1:6000); Alexa-568-conjugated goat anti-mouse/rabbit and Alexa-488-conjugated goat anti-rabbit/mouse secondary antibodies (Molecular Probes, 1:300); Cy5 goat-anti-rabbit secondary antibody (Molecular Probes, 1:300); rhodamin-conjugated phalloidin (Invitrogen, 1:200); DAPI (49,69-diamidino-2-phenylindole, Sigma; 0.1 mg/ml, 5 min incubation), and in situ cell death detection kit (Roche). Images were captured by either a Zeiss Imager Z1 equipped with an ApoTome system or a Zeiss Meta 510 confocal microscope. All images were processed in Adobe Photoshop and Canvas. FACS Guts were dissected in PBS and sectioned. The sectioned guts were incubated in PBS with 0.5% trypsin (Invitrogen), 1 mM EDTA and 10 mg/ml bisBenzimide H33342 trihydrochloride (Hoechst33342, Sigma) at room temperature for 2.5 h on a rotator, and vortexed every 30 min. The undissociated cells were removed by 70 mm cell strainer. The dissociated cells were analyzed by BD FACSAria II. FCS Express 4 were used to analyze the cell cycle profiling.

Acknowledgements We are grateful to Drs. Mark Krasnow, Akira Chiba, Jean-Rene´ Huynh, Martin-Bermudo, Alana O’Reilly, M. Piefer, Shigeo Hayashi, Stefan Baumgartner, Doug Harrison, Alfonso Marinaz-Arias, and Dick Na€ssel for fly stocks and antibodies, the Bloomington Stock Center, Developmental Studies Hybridoma Bank (DSHB), Vienna Drosophila RNAi Center (VDRC) and National Institute of Genetics Fly Stock Center (NIG-Fly) for fly stocks and reagents, and Yi Huang and Dr. Wenhui Li for assistance with FACS analysis. This work was supported by the Chinese Ministry of Science and Technology through 973 (2011CB12700) and 863 (2007AA02Z1A2) grants.

Mosaic analysis

Appendix A. Supporting information

In all experiments, posterior midguts were analyzed. MARCM clones were induced by heat shock treatment of 3–5 day-old females with appropriate genotypes at 37 1C running water bath for 30 min. Flies were dissected and stained at day 4, 7, and 14 after clone induction. For ectopic expression by UAS/GAL4ts system, the fly cross was done at 18 1C, then the 3–5 day-old females with appropriate genotypes were shifted to 29 1C to induce transgene expression, then analyzed at day 5 and 14. To induce apoptosis in esgþ cells, flies of genotype esgGAL4 ts/UAS-rpr-mts were shifted

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2013.01.032.

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