Journal of Molecular Cell Biology Advance Access published online on September 30, 2009
Journal of Molecular Cell Biology, doi:10.1093/jmcb/mjp028
Paracrine Unpaired Signaling through the JAK/STAT Pathway Controls Self-renewal and Lineage Differentiation of Drosophila Intestinal Stem Cells
1 National Institute of Biological Sciences, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China
2 College of Life Sciences, Beijing Normal University, Beijing 100875, China
* Correspondence to: Rongwen Xi, E-mail: xirongwen{at}nibs.ac.cn
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Abstract |
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Drosophila and mammalian intestinal stem cells (ISCs) share similarities in their regulatory mechanisms, with both requiring Wingless (Wg)/Wnt signaling for their self-renewal, although additional regulatory mechanisms are largely unknown. Here we report the identification of Unpaired as another paracrine signal from the muscular niche, which activates a canonical JAK/STAT signaling cascade in Drosophila ISCs to regulate ISC self-renewal and differentiation. We show that compromised JAK signaling causes ISC quiescence and loss, whereas signaling overactivation produces extra ISC-like and progenitor cells. Simultaneous disruption or activation of both JAK and Wg signaling in ISCs results in a stronger ISC loss or a greater expansion of ISC-like cells, respectively, than by altering either pathway alone, indicating that the two pathways function in parallel. Furthermore, we show that loss of JAK signaling causes blockage of enteroblast differentiation and reduced JAK signaling preferentially affects enteroendocrine (ee) cell differentiation. Conversely, JAK overactivation produces extra differentiated cells, especially ee cells. Together with the functional analysis with Notch (N), we suggest two separate roles of JAK/STAT signaling in Drosophila ISC lineages: it functions upstream of N, in parallel and cooperatively with Wg signaling to control ISC self-renewal; it also antagonizes with N activity to control the binary fate choice of intestinal progenitor cells.
Keywords: intestinal stem cell, Drosophila, the JAK/STAT pathway, Wingless signaling, self-renewal and differentiation, enterocyte, enteroendocrine cell
Received July 21, 2009; Revised August 24, 2009; Accepted September 1, 2009
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Introduction |
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Adult stem cells reside in many tissues and play a critical role in maintaining tissue homeostasis throughout life. The microenvironments or niches that harbor and control stem cells represent a common mechanism for stem cells' long-term maintenance, proliferation and differentiation (Morrison and Spradling, 2008). Therefore, studying the regulatory interactions between stem cells and their niches is critical for understanding how homeostasis is controlled under normal conditions and how it goes awry in diseases. In mammals, intestinal stem cell (ISC) at the crypt bottom maintains homeostasis of the intestinal epithelium, one of the most rapid cycling tissues, yet stem cell and niche interaction in the intestinal epithelium is poorly understood (van der Flier and Clevers, 2009).
Drosophila midgut has recently emerged as an attractive system to study epithelial homeostasis control in the intestine (Casali and Batlle, 2009). A single-layered intestinal epithelium in the midgut is maintained by multipotent midgut ISCs that reside basally on a thin layer of basement membrane right beneath the surrounding visceral muscles and steadily produce progenitor cells named enteroblasts (EBs), which will undergo further differentiation into either absorptive enterocytes (ECs) or secretary enteroendocrine (ee) cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006) (Figure 1A). Besides structural and lineage similarities to the mammalian intestine, Drosophila midgut also shares certain genetic control mechanisms for ISC self-renewal and lineage differentiation. Paracrine Wingless (Wg)/Wnt signal from muscle cells directly activates a canonical Wnt signaling cascade to regulate Drosophila ISC maintenance, proliferation and differentiation (Lin et al., 2008). Wnt signaling is known as a key player in mammalian ISCs (van der Flier and Clevers, 2009). In Drosophila ISC lineage, Notch (N) activation is critical for lineage differentiation and disruption of N function in ISC results in the production of ISC-like tumors due to differentiation blockage (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). The N ligand Delta (Dl) is specifically expressed in the ISCs with variable expression levels. It is suggested that ISCs with high Dl expression will activate N at a high level in EBs to promote their differentiation toward EC fate, whereas ISCs with low Dl expression will activate N at a low level in EBs to allow their differentiation toward ee cell fate (Ohlstein and Spradling, 2007). Therefore, differential levels of N activity control the binary switch of the progenitor cells toward either EC or ee cell fate. However, because N is required for both EC and ee cell differentiation, additional mechanisms are probably involved in determining this binary fate choice. In mammals, the N pathway also controls the absorptive versus secretary fate choice (van der Flier and Clevers, 2009), indicating that genetic control of intestinal lineage differentiation could also be conserved from Drosophila to mammals.
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Our previous observations indicate potential existence of additional mechanisms in controlling ISC self-renewal, in addition to Wg signaling. Disrupting Wg pathway activity in ISCs causes mild decline of ISC number over time, whereas pathway hyperactivation only induces limited expansion of ISCs and progenitors without complete blockage of terminal differentiation (Lin et al., 2008). Because stem cell self-renewal is commonly governed by multiple signaling pathways, we performed a genetic screen for other signaling pathways required for ISC maintenance and identified the JAK/STAT pathway as another important signaling cascade in controlling ISC maintenance, proliferation and differentiation. The JAK/STAT pathway is an evolutionarily conserved signaling cascade from Drosophila to vertebrates that transduces cytokine signals (Rawlings et al., 2004). In Drosophila, the pathway activation is triggered by Unpaired (Upd) (Harrison et al., 1998) and other two Upd-like molecules (Agaisse et al., 2003; Gilbert et al., 2005; Hombria et al., 2005). The signal is transduced through the membrane receptor Domeless (Dome) (Brown et al., 2001; Chen et al., 2002), the receptor-associated Janus Kinase Hopscotch (Hop) (Binari and Perrimon, 1994) and the downstream transcription factor STAT92E (Hou et al., 1996; Yan et al., 1996). The pathway has important functions in many biological processes in Drosophila, including hematopoiesis, immunity and stem cell maintenance (Arbouzova and Zeidler, 2006; Gregory et al., 2008).
In this study, we show that the JAK/STAT pathway functions cooperatively and in parallel with Wg signaling to control ISC self-renewal and functions antagonistically with N activity to modulate the binary fate decision during progenitor cell differentiation. Our study reveals a novel player in stem cell–niche interaction and downstream lineage differentiation in the Drosophila intestine, which may provide important implications for ISC regulation and intestinal dysfunction in mammals.
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Results |
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The JAK/STAT pathway activity in the Drosophila midgut
In a search for additional signaling pathways involved in ISC regulation, we identified another signaling molecule encoded by upd, the major ligand for the JAK/STAT pathway in Drosophila (Harrison et al., 1998) that was also specifically expressed in the ISC niche. A lacZ enhancer trap of upd showed specific expression of nuclear β-galactosidase in the inner circular muscles and outer longitudinal muscles, but not in the epithelial cells (Figure 1B). Similarly, immunostaining with antibodies against Upd also showed specific Upd expression in muscle cells (Figure 1C). Upd expression in the muscular niche suggests a possible role of Upd in regulating ISCs. It could function as an autocrine signal in muscle cells, or as a paracrine signal to regulate epithelial cells. We thus examined the expression of a pathway activation reporter in the midgut to determine where the pathway is activated. 10 x STAT-GFP is a GFP reporter with 10 copies of STAT-binding sites at the promoter, which serves as a reliable marker for STAT92E activation in vivo (Bach et al., 2007). GFP signal was detected in muscle cells as well as a subpopulation of diploid cells in the epithelium (Figure 1F). Co-staining with antibodies against Dl, an ISC-specific marker, and Prospero (Pros), an ee cell-specific marker, showed that GFP was specifically expressed in ISCs and EBs, but was not detectable in ee cells and ECs in the epithelium (Figure 1D). We also examined dome-lacZ (dome-MESO), a lacZ enhancer trap of dome (Brown et al., 2001). Dome is the receptor for Upd signaling, whose transcription is also positively regulated by the pathway activity. dome-lacZ expression was detected in muscle cells as well, albeit at low levels (Figure 1G). In the epithelium, it was also specifically detected in ISCs and EBs but not in ECs or ee cells (Figure 1E). These observations indicate that Upd, which is produced from the surrounding muscle cells, could traverse through the basement membrane, reach ISCs and activate the downstream signaling cascade in ISCs in a manner similar to Wg molecules.
The ligand Upd is required for ISC maintenance and proliferation
We further examined the requirement of upd in ISC regulation. upd is an essential gene for viability and to study the function of upd, we took advantage of hypomorphic alleles of upd, and made transheterozygous upd4 (or named updYM55)/ossiscG20 and upd4/oss flies that could survive to adulthood (Harrison et al., 1998). The percentage of Dl+ ISCs from total epithelial cells was 9.8 ± 0.96% (intestines examined, n = 5) in upd4/+ intestines, 5.6 ± 0.38% (n = 4) in upd4/ossiscG20 intestines and 7.0 ± 0.54% (n = 4) in upd4/oss intestines (Figure 2A–C), suggesting that reduced upd function caused a decline of ISC population. The percentage of Phospho-histone 3 (PH3)-labeled ISCs was 6.8 ± 0.65% (intestines examined, n = 5) in upd4/+ , 4.0 ± 0.60% (n = 5) in upd4/ossiscG20 and 4.3 ± 0.56% (n = 7) in upd4/oss intestines (Figure 2D), suggesting that ISCs were less proliferative in upd mutant intestines. TUNEL labeling for apoptotic cells showed no obvious increase of ISC death in upd4/ossiscG20 (0/118) and upd4/oss intestines (0/155). Taken together, these data suggest that upd is required for maintenance and proliferation of ISCs, but not for their survival.
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Canonical JAK/STAT signaling components are cell-autonomously required for ISC maintenance
We next determined cell-autonomous requirements of upd, dome, hop and stat92E in ISCs by generating GFP-marked mutant ISC clones using the MARCM technique (Lee et al., 2000), and examining their maintenance and proliferation over time, as previously described (Lin et al., 2008). The initially GFP-marked normal or mutant ISCs were generated by heat shock-induced mitotic recombination in flies of appropriate genotypes. As a control, the marked wild-type ISCs were able to generate and maintain clusters of GFP-labeled clones containing ISCs, progenitors and differentiated ECs and ee cells (Figure 3A and data not shown). These wild-type ISC-derived clones showed a slow ISC turnover rate over time, with 89.0% of GFP clones still containing ISCs 2 weeks after clone induction (Figure 3I), consistent with previous observations (Lin et al., 2008). upd4 is a strong or genetic null allele of upd (Harrison et al., 1998), and upd4 homozygous ISC-containing clones behaved similar to the wild-type ones, as the mutant ISCs had a similar maintenance rate as the wild-type ones and properly differentiated cells were present within the mutant clones (Figure 3B and I). These observations are consistent with a non-cell-autonomous role of upd in regulating ISCs. To exclude the possibility that two additional ligands, Upd2 and Upd3, may have redundant functions in ISCs, we generated homozygous Df(1)os1A mutant ISC-derived clones. Df(1)os1A is a chromosome deletion allele, in which all the coding regions of upd, upd2 and upd3 are deleted and therefore it is considered as a null allele for all JAK signaling ligands (Brown et al., 2003). Homozygous Df(1)os1A mutant ISC-containing clones also behaved similar to the wild-type ones, with no obvious maintenance, proliferation or differentiation defects (Figure 3C and I), further suggesting that under normal conditions, JAK/STAT ligands do not function as autocrine signals in ISCs. Strikingly, ISCs that were homozygous for domeG0468, domeG0405, hopC111, hopM4 or stat6346, all representing loss-of-function alleles for cognate genes (Binari and Perrimon, 1994; Hou et al., 1996; Brown et al., 2001), displayed a rapid ISC loss phenotype over a short time period, with only 31.3%, 40.1%, 25.5 %, 20.7% and 49.8% ISC clones maintained 2 weeks after clone induction, respectively, for each allele (Figure 3D–F and I). In addition, many of these mutant ISC-containing clones contained smaller number of cells, which is likely caused by reduced proliferation of the mutant ISCs. We thus performed pulsed Bromodeoxyuridine (BrdU) labeling experiment to evaluate ISC division rate. As a control, the wild-type ISCs had 30.5% (n = 236) labeled with BrdU at 1 week after clone induction. In contrast, domeG0468, domeG0405, hopC111and hopM4 mutant ISCs had only 14.3% (n = 28), 19.0% (n = 21), 10.7% (n = 28) and 20.0% (n = 15) labeled, respectively for each allele, suggesting that the mutant ISCs were underproliferative. Using a FLP-OUT system (Struhl and Basler, 1993), we also expressed a dominant negative form of dome (domeDN) in ISC clones marked by GFP. Forced domeDN expression also led to rapid loss of ISCs (Figure 3H), further supporting a cell-autonomous requirement of JAK/STAT activation in maintaining ISCs. We conclude that the canonical JAK/STAT pathway components, including Dome, Hop and STAT92E, are cell-autonomously required for ISC maintenance and proliferation. These observations suggest a model similar to paracrine Wg signaling, Upd is another paracrine signaling that is produced from the muscular niche and directly acts on ISCs, where it triggers a canonical JAK/STAT signaling cascade to regulate the maintenance and proliferation of ISCs.
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JAK/STAT and Wingless signaling function in parallel for ISC maintenance and proliferation
Because Wg signaling has a similar role in regulating ISC maintenance and proliferation, we further investigated the genetic relationships between JAK/STAT and Wg signaling in regulating ISCs. We asked whether disrupting both pathway activities could produce a stronger phenotype than disrupting either pathway alone. In Drosophila, Wg signal is transduced by the Frizzled and LRP family of membrane receptors and the scaffold protein Dishevelled (Dsh), followed by the inhibition of the Axin/GSK3/APC complex and the stabilization of Armadillo (Arm)/β-catenin. Arm then translocates to nucleus to regulate transcription of target genes (Logan and Nusse, 2004). We have previously shown that Wg signaling-compromised ISCs, including arm mutant ISCs, show a slow turnover phenotype, with 55–85% ISC-containing clones still maintained when examined at 2 weeks after clone induction (Lin et al., 2008). We generated hop arm and dome arm double-mutant ISC clones and studied their behavior over time. The double-mutant ISC clones showed a stronger ISC loss phenotype than single-mutant ISC clones, with only 8.8% and 10.6% labeled ISCs maintained after 2 weeks, respectively, for arm3 domeG0468 and arm3 hopC111 mutant ISCs (Figure 3G and I). In addition, the double-mutant ISCs showed more severe decline of BrdU labeling rates: 9.5% (n = 24) for arm3 domeG0468 mutant ISCs and 8.3% (n = 21) for arm3 hopC111 mutant ISCs, compared with either hop or arm single-mutant ISCs (Lin et al., 2008). The ability to enhance each other's phenotype in double mutants indicates that JAK/STAT and Wg signaling function cooperatively and in parallel in regulating ISC maintenance and proliferation.
The JAK/STAT pathway is essential for intestinal lineage differentiation
We noticed that in dome and hop homozygous mutant ISC-derived clones, polyploid EC or Pros+ ee cell was much less frequently observed (Figure 3D and E; Table 1). Instead, almost all non-ISC cells in the mutant clones were EB-like, as they displayed small diploid nuclear sizes and did not express either Dl or Pros (Figure 3D and E). Similar observations were found in domeDN overexpression clones (Figure 3H). To further confirm that the mutant cells were EB-like cells, we utilized a temperature-sensitive Gal4/UAS system (Brand and Perrimon, 1993; McGuire et al., 2003) to express UAS-domeRNAi and UAS GFP in ISCs and progenitor cells with an esgGal4 driver, which drives UAS-transgene expression specifically in ISCs and EB cells (Micchelli and Perrimon, 2006). dome RNAi also resulted in reduced number of Dl+ ISCs. It also produced many Dl– GFP+ small diploid cells (Figure 4A and A'), indicating that these small diploid cells are EB-like, which is also consistent with the observation by a recent study (Jiang et al., 2009). These EB-like cells also do not divide, as they do not incorporate BrdU in the BrdU labeling assay (data not shown). Furthermore, Pdm-1, an EC-specific marker (Lee et al., 2009), was not expressed in these mutant cells (Figure 4B and 4B'). Because N is essential for EB differentiation, we further examined the expression of an N activation reporter Su(H)m8-lacZ (Furriols and Bray, 2001) in these mutant cells. Normally, Su(H)m8-lacZ is not expressed in ISCs, but is expressed in EBs where N is activated (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Many of these EB-like cells in hop mutant clones showed expression of Su(H)m8-lacZ (Figure 4C and 4C'), indicating that the defective differentiation of the JAK signaling mutant cells is not caused by failed activation of N. Taken together, these data indicate that JAK signaling-compromised ISCs are differentiation-defective: they are blocked at the EB-like stage and could not differentiate further.
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Therefore, JAK/STAT signaling may have a critical role in promoting EB differentiation toward both EC and ee cell directions. Polyploid ECs could be frequently observed in Stat92E06346 mutant ISC-containing clones (86.6%) (Table 1), likely because this allele does not completely eliminate Stat92E gene function. Similarly, in domeDN FLP-OUT clones, ECs were also frequently observed (75.0%). However, ee cell differentiation seemed to be particularly affected in DomeDN or Stat92E06346 mutant ISC-containing clones, as only 7.1% and 9.0% mutant clones, respectively, for each genotype, contained ee cells, compared with 44.8% for wild-type clones (Table 1). These data indicate that although JAK/STAT signaling is essential for both EC and ee cell differentiation, a high level of pathway activity is particularly critical for ee cell differentiation.
JAK signaling activation causes a limited expansion of the ISC population but a dramatic increase of ee cell population
To test whether JAK/STAT signaling activation is sufficient to promote ISC self-renewal, we again utilized the temperature-sensitive Gal4/UAS system to express upd or hop in ISCs and progenitor cells with the esgGal4 driver. Normally, individual ISCs are scattered along the basement membrane of the epithelia, and are marked by Dl and GFP expression (Figure 5A). We have previously shown that forced activation of Wg signaling by expressing an active form of Arm was able to cause weak expansion of ISC-like and EB-like cells, but wg overexpression seemed to be able to produce a stronger phenotype (Lin et al., 2008). After testing several independent UAS-wg lines and rebalancing the original line, we found that wg overexpression also produced a weak phenotype similar to that caused by Arm overexpression (Figure 5B and C) and the originally observed stronger phenotype was possibly contributed by certain genetic background. Forced expression of upd or hop also caused weak expansion of ISC-like cells (Dl+, GFP+) and EB-like cells (Dl–, GFP+) (Figure 5D–E). Unlike Wg signaling activation, however, overexpression of upd or hop also led to a dramatic increase of ee-like cells (Pros+, GFP–) in the epithelium (Figure 5D–E'). Many of these Pros+ cells also had Tachykinin or Allatostatin expression (data not shown), each marking different subtypes of ee cells (Ohlstein and Spradling, 2006), indicating that these extra ee-like cells could be terminally differentiated. The percentages of ee cells and ECs in total epithelial cells were 27.2 ± 4.85% and 44.2 ± 7.62% (intestines examined, n = 5), respectively, in upd overexpressed intestines and 18.4 ± 3.07% and 52.7 ± 7.24% (n = 5) in hop overexpressed intestines, compared with 6.9 ± 0.47% and 61.7 ± 1.38% (n = 8) in the wild-type intestines (Figure 5F), showing that there were 3- to 4-fold increases of the percentage of ee cells in the epithelium. These data suggest that JAK/STAT signaling activation could generate two separate effects on the ISC lineage: promoting ISC self-renewal and promoting progenitor cell differentiation, preferentially toward ee cell fate.
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Co-activation of JAK and Wg signaling synergistically promotes ISC self-renewal
We next tested the effects of simultaneous activation of JAK/STAT and Wg signaling in ISCs by expressing UAS-hop and UAS-arm
N or UAS-upd and UAS-armN. Simultaneous activation of both pathways caused dramatic enhancement of ISC-like cell (Dl–, GFP+) accumulation than by activating either pathway alone (Figure 6). Co-expression of UAS-upd and UAS-armN also frequently produced spherical-shaped tumor-like cell masses that were filled with ISC-like cells (Dl+, GFP+) and progenitor cells (Dl–, GFP+) (Figure 6B and B'). Consequently, the intestinal tract frequently displayed disorganized morphology at these regions (Figure 6B and B'). This ISC-like cell accumulation was accompanied by the reduced population of ee cells and ECs at these regions (Figure 6), suggesting that the production of these ISC-like and progenitor cells is at the expense of differentiated cells and is a consequence of both enhanced ISC self-renewal and blockage in differentiation. Activation of both pathways, however, still could not completely inhibit ISC differentiation, as EB-like cells, ECs and ee cells were still observed. This raises a possibility that additional regulators could be involved in ISC self-renewal. We suggest that JAK/STAT, Wg and potentially other regulators function together to control the self-renewal of ISCs in the Drosophila midgut (Figure 7D).
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Antagonistic activities of JAK and Notch control binary fate choice of intestinal progenitors
Previous studies suggest that N functions downstream of Wg signaling in regulating ISC self-renewal (Lin et al., 2008). To test the epistatic relationships between JAK/STAT signaling and N, we generated dome N double-mutant ISCs and stat92E mutant ISCs with the expression of N RNAi. Similar to N mutation alone (Figure 7A), Dl+ ISC-like tumors were readily observed in dome N as well as in stat92E N mutant clones (Figure 7B and C), suggesting that N is epistatic to JAK/STAT signaling in ISC self-renewal. ee cell-like tumor, which is commonly observed in N mutant clones (Figure 7A), however, was rarely observed in the double-mutant clones (Figure 7B and C), suggesting that JAK signaling is required for N mutation-induced ee cell-like tumor formation, which further supports the notion that JAK/STAT signaling activity favors ee cell differentiation from progenitor cells. Together with a role of N in favoring EC over ee cell differentiation, we propose that Nhigh and JAKlow allow EB differentiation into EC, whereas Nlow and JAKhigh allow EB differentiation into ee cell, and antagonistic activities of N and STAT signaling control the binary decision between EC and ee cell fate (Figure 7D).
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Discussion |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and methodsFundingReferences |
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In this study, we identified two novel and separate roles of JAK/STAT signaling in ISC lineages in the Drosophila midgut. It functions as an important paracrine signaling mechanism that acts cooperatively and in parallel with Wg signaling to regulate ISC self-renewal. It also plays a critical role in promoting terminal differentiation from epithelial progenitors, preferentially toward the ee cell fate. Because many aspects of the genetic control in the intestine are conserved from Drosophila to vertebrate, our study may provide important implications for the understanding of ISC self-renewal and differentiation in mammals and humans.
Our previous studies revealed a muscular niche for the Drosophila midgut ISCs where Wg signal is produced (Lin et al., 2008). In this study, we have identified another signal molecule that is also produced from the muscular niche and acts directly on ISCs, where it activates the JAK/STAT pathway to control ISC maintenance and proliferation. Therefore, ISCs are controlled by multiple signals from the niche. Importantly, our genetic analysis indicates that these two pathways function cooperatively and in parallel for ISC maintenance and self-renewal. The cooperative function between these two pathways for ISC self-renewal may help to explain the limited sufficiency of either Wg or JAK/STAT signaling activation alone in promoting ISC self-renewal. Wg pathway activation through overexpression of Wg or an active form of Arm only causes a limited increase of Dl+ esg+ ISC-like cells, similar to that caused by JAK signaling activation. In addition, functional loss of shaggy, a negative regulator of Wg signaling, only causes ISC overproliferation without any apparent effect on ISC self-renewal (Lin and Xi, 2008; Lin et al., 2008). Similarly, Wg pathway activation by mutations in adenomatous polyposis coli (Apc) or Axin promotes ISC proliferation but does not seem to affect ISC self-renewal (Lee et al., 2009). However, co-activation of Wg and JAK signaling dramatically increases Dl+ esg+ ISC-like cells, indicating that ISC self-renewal requires simultaneous activation of both pathways. It should be noted that co-activation of both pathways still could not completely block the differentiation of all ISCs, probably because the levels of transgene expression are variable, or there could be additional players required for ISC self-renewal.
Previous epistatic analysis suggests that Wg signaling may act upstream of N in regulating ISC self-renewal (Lin et al., 2008). Similarly, N is also epistatic to JAK/STAT in regulating ISC self-renewal, as N and JAK/STAT double-mutant ISCs produce ISC-like tumors that mimics the outcomes by N mutation alone, indicating that Wg and JAK signaling function upstream of N. Possibly, Wg and JAK signaling could control N activity by regulating Dl expression directly or indirectly in ISCs. In either N dome or N dsh double-mutant ISCs, however, Dl expression was maintained, indicating that Wg and JAK signaling are not absolutely required for Dl expression at least when N is inactivated. It would be important to further dissect how JAK/STAT and Wg signaling interact with the Dl-N regulatory circuitry in controlling ISC self-renewal and differentiation.
In addition to midgut ISCs, STAT92E is also specifically activated in Drosophila hindgut stem cell population (Takashima et al., 2008), indicating a similar role of JAK/STAT signaling in hindgut ISCs. Several recent studies suggest that Drosophila JAK/STAT signaling may also mediate injury- or infection-induced ISC proliferation and regeneration (Amcheslavsky et al., 2009; Buchon et al., 2009; Cronin et al., 2009; Jiang et al., 2009), indicating that under stress conditions, niche signals could be regulated by novel mechanisms to promote ISC division and tissue regeneration.
This study also provides additional insights underlying cell fate determination from progenitor cells during intestinal lineage differentiation. Our gain and loss of function studies suggest that, although JAK signaling is essential for the differentiation of both EC and ee cells, a high level of JAK activation favors differentiation toward the ee cell fate, whereas a low level of JAK activation favors differentiation toward EC. These observations indicate that JAK activity reversely correlates with N activity and antagonistic activities of JAK and N control the binary choice between EC and ee cell fate from intestinal progenitor cells. Interestingly, antagonistic activities of JAK and N in specifying cell fates have been implicated in a number of other developmental processes. For example, antagonistic activities of N and JAK control the choice between polar and stalk cell fate in Drosophila oogenesis (McGregor et al., 2002). In the polar cells, N activation blocks nuclear translocation of STAT92E and consequently inhibits JAK signaling activation (Assa-Kunik et al., 2007). In the Drosophila eye, STAT92E represses the expression of the N ligand Serrate to inhibit N activation in the dorsal eye to control eye growth (Flaherty et al., 2009). It would be interesting to investigate whether N and JAK directly antagonize each other in the Drosophila intestine and to identify their respective target genes that promote different directions of cell differentiation. In addition to signaling pathways, cell adhesion molecules may be also involved in regulating this binary fate choice. Sustained membrane contact between ISC and EB through E-cadherin-mediated homophilic adhesion could facilitate N activation in EBs to promote their differentiation into ECs rather than ee cells (Maeda et al., 2008). We suggest that a signaling output from regulatory interactions among N, JAK/STAT signaling, cell adhesion molecules and other environmental inputs may ultimately determine the binary fate choice of an EB in the Drosophila midgut.
Recent studies in mammals also indicate that in addition to Wnt signaling, there could be additional regulators controlling ISC fate. ISCs at the crypt bottom of the mouse small intestine specifically express Lgr5 and Asl2, both are Wnt target genes (van der Flier et al., 2009). There are also other genes that are not Wnt targets but also specifically expressed in ISCs, such as Olfm4. Asl2 is essential for ISC self-renewal, but its overexpression only induced limited expansion of Olfm4+ cells within the crypt region (van der Flier et al., 2009). In addition, Wnt signaling activation in ISCs by APC deficiency causes adenoma development and in these adenoma cells, only a small fraction of these cells maintains Lgr5 expression (Barker et al., 2009). These observations indicate that additional regulators are required in addition to Wnt signaling to define ISC fate in the mouse small intestine. The potential role of the mammalian JAK/STAT signaling in governing ISC fate is not clear, but JAK/STAT signaling has been implicated in facilitating inflammation-associated intestinal tumorigenesis and might also promote tumorigenesis initiated by APC deficiency (Baltgalvis et al., 2008; Bollrath et al., 2009; Grivennikov et al., 2009). Thus, our genetic analysis of the Drosophila ISC niche may also provide insights underlying ISC self-renewal, homoeostasis control and tumorigenesis in mammals and humans.
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Materials and methods |
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Fly strains
Flies were maintained at 25°C and cultured on standard media, unless otherwise stated. The following fly stocks were used in this study and their description can be found in Flybase or as otherwise specified: upd-lacZ; upd4; ossiscG20; oss; Df(1)os1A; domeG0468; domeG0405; hopC111; hopM4; stat92E06346; arm3; 10 x STAT92E-GFP; dome-MESO(dome-lacZ); UAS-dome
CYT (UAS-domeDN); UAS-wingless; UAS-upd(PK9); UAS-hop(3W); UAS-armN (1-155) (Zecca et al., 1996); Su(H)m8-lacZ; esg-Gal4,UAS-GFP; N264–40; UAS-Notch-dsRNA(BSC); UAS-dome-dsRNA (VGRC).
Mosaic analysis
In all experiments, posterior midgut was the region analyzed. For MARCM clones, fly crosses were made to generate the following adults of genotypes: hsflp122/+ , tub-gal80 FRT19A/FRT19A; Act-Gal4, UASGFP/+ ; hsflp122/+ , tub-gal80 FRT19A/upd4 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/+ , tub-gal80 FRT19A/domeG0468 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/+ , tub-gal80 FRT19A/hopC111 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/+ ; Act-Gal4, UAS-GFP/+ ; Tub-Gal80 FRT82B/stat92E06346 FRT82B; hsflp122/+ , tub-gal80 FRT19A/arm3domeG0468 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/+ , tub-gal80 FRT19A/arm3hopC111 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/+ , tub-gal80 FRT19A/N264-40domeG0468 FRT19A; Act-Gal4, UAS-GFP/+ ; hsflp122/UAS-Notch dsRNA; Act-Gal4, UAS-GFP/+ ; Tub-Gal80 FRT82B/stat6346 FRT82B. Clones were induced by 1 h heat shock treatments of 3–5-day-old females in a 37°C running water bath. To overexpress domeDN using the FLP-OUT cassette, the following adults of genotype were generated: hsflp122/+ ; Act5C > y > Gal4, UAS-GFP/UAS-domeN. Flies were heat-shocked at 37°C for 1 h and were dissected and stained 2 weeks after clone induction.
Gal4ts/UAS system
For overexpression experiments using esgGal4 and Tub-gal80ts, fly crosses were done in 18°C, and newly enclosed flies of appropriate genotypes were shifted to 29°C for 2 weeks before dissection. Fresh food with yeast paste was replaced every 2 days. The middle one-third region of the posterior midgut was analyzed in all overexpression experiments.
Immunohistochemistry
The immunostaining protocol used in this study has been described previously (Lin et al., 2008). Briefly, intestines were dissected in Grace's media at room temperature, and fixed in a 1:1 (v/v) mixture of 4% formaldehyde and n-heptane for 15 min. The lower aqueous phase of the mixture was removed and replaced by an equal volume of methanol. Subsequently, the tubes were hand shaked vigorously for 30 sec. Samples were then washed twice in methanol and gradually rehydrated in PBT (10 mM NaH2PO4/ Na2HPO4, 175 mM NaCl, pH 7.4, plus 0.1% Triton X-100). After block in 5% normal goat serum in PBT for 1 h, samples were incubated in appropriate primary antibodies at 4°C overnight. Secondary antibodies were incubated for either 2 h at room temperature or overnight at 4°C. Samples were mounted in 70% glycerol. The following antisera or dyes were used: mouse anti-Dl antibody [Developmental Studies Hybridoma Bank (DSHB); 1:100]; rabbit anti-Upd antibody (a gift from Doug Harrison, 1:1 000); mouse anti-Pros antibody (DSHB, 1:300); rabbit anti-Pdm1 (a gift from Xiaohang Yang, 1:1000); mouse anti-Allatostatin (DSHB, 1:10); rabbit anti-tachykinin (a gift from Dick Nässel, 1:3000); rabbit anti-BrdU antibody (1:300); rabbit anti-phospho-Histone H3 antibody (Upstate, 1:1000); rabbit polyclonal anti-β-gal antibodies (Cappel, 1:6000); Alexa-568-conjugated goat anti-mouse/rabbit and Alexa-488-conjugated goat anti-rabbit/mouse secondary antibodies (Molecular Probes, 1:300); rhodamine-conjugated Phalloidin (Molecular Probes, 1:500); 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 Illustrator.
BrdU incorporation
Intestines were dissected in PBS and incubated in 100 µg/ml BrdU (Sigma) in PBS for 1 h at room temperature in the dark. After rinsed with PBS and then fixed as staining other antibodies, the guts were treated with DNase I for 30 min at 37°C. The reaction was stopped by washing samples with PBT. Subsequent immunostaining procedures were as described above.
Graphs and calculations
The percentages of ISCs (Figure 2C), ee cells and ECs (Figure 5F) were calculated as the number of ISCs, ee cells or ECs divided by the total number of epithelial cells per midgut; the percentage of pH3-positive ISCs (Figure 2D) was calculated as the number of pH3-positive ISCs divided by the total number of ISCs per midgut. Statistical analysis was done using Student's t-test. The time course clonal analysis (Figure 3I) was performed as previously described (Lin et al., 2008). Basically, we quantified the number of ISC-containing clones per midgut at Days 4, 7 and 14 after clone induction, then divided this number by the average number of ISC-containing clones at Day 4 to get the percentage of ISC-containing clones maintained at each time points. Ten to 20 intestines were examined for each genotype at each time point. The graphs were made using windows Prism 4 software.
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TopNotesAbstractIntroductionResultsDiscussionMaterials and methodsFundingReferences |
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This work was supported by an ‘863’ grant (2007AA02Z1A2 to R.X.) from the Chinese Ministry of Science and Technology.
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Acknowledgements |
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We thank Erika Bach, Shigeo Hayashi, James Castelli-Gair Hombria, Acamio Gonzalez-Reyes, Doug Harrison, Hong Luo, Alfonso Marinaz-Arias, Gary Struhl, Sarah Bray, Xiaohang Yang, Dick Nässel, Zhaohui Wang, Ting Xie, the Bloomington Stock Center, Developmental Studies Hybridoma Bank (DSHB) and Vienna Drosophila RNAi Center (VDRC) for reagents and members of the Xi laboratory for comments and discussions.
Conflict of interest: none declared.
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These authors contributed equally to this work. 
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