Article

Paracrine Unpaired Signaling through the JAK/STATPathway Controls Self-renewal and LineageDifferentiation of Drosophila Intestinal Stem CellsGuonan Lin1,†, Na Xu1,2,†, and Rongwen Xi1,*

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@nibs.ac.cn

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 sig-

naling 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

IntroductionAdult stem cells reside in many tissues and play a critical role in

maintaining tissue homeostasis throughout life. The microenvir-

onments or niches that harbor and control stem cells represent

a common mechanism for stem cells’ long-term maintenance, pro-

liferation and differentiation (Morrison and Spradling, 2008).

Therefore, studying the regulatory interactions between stem

cells and their niches is critical for understanding how homeosta-

sis 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 inter-

action 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 struc-

tural and lineage similarities to the mammalian intestine,

Drosophila midgut also shares certain genetic control mechan-

isms 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 main-

tenance, 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) acti-

vation 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 specifi-

cally expressed in the ISCs with variable expression levels. It is

suggested that ISCs with high Dl expression will activate N at a

†These authors contributed equally to this work.

Received July 21, 2009. Revised August 24, 2009. Accepted September 1, 2009.# The Author (2009). Published by Oxford University Press on behalf of Journal of

Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

doi:10.1093/jmcb/mjp028 Journal of Molecular Cell Biology (2009), 1–13 j 1

Journal of Molecular Cell Biology Advance Access published September 30, 2009

Figure 1 Upd expression and JAK/STAT signaling activity in the midgut. (A) A diagram showing a cross-section of the midgut epithelium. (B) A

cross-section stained with upd-lacZ (green) and Phalloidin (red). (C) A cross-section stained with anti-Upd (green) and anti-Dl antibody (red).

Note that Upd is enriched in the muscle cells. (D) A superficial section through the epithelium stained with 10 � STAT92E-GFP (green), anti-Dl

antibody (red, membrane) and anti-Pros antibody (red, nucleus). Note that GFP was specifically expressed in the ISCs and EBs. (E) dome-lacZ

(green) was also specifically expressed in ISCs and EBs. (F) A cross-section showing weak 10XSTAT-GFP (green) expression in the muscle cells.

(G) A cross-section showing weak dome-lacZ (green) expression in the muscle cells. DAPI (49,69-diamidino-2-phenylindole) nuclear staining

was shown in blue. Scale bars, 10 mm. ISC, intestinal stem cell; EB, enteroblast; EC, enterocyte; ee, enteroendocrine cell.

2 j Journal of Molecular Cell Biology Lin et al.

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 sec-

retary fate choice (van der Flier and Clevers, 2009), indicating that

genetic control of intestinal lineage differentiation could also be

conserved from Drosophila to mammals.

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 hyperacti-

vation 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 ident-

ified the JAK/STAT pathway as another important signaling

cascade in controlling ISC maintenance, proliferation and differen-

tiation. The JAK/STAT pathway is an evolutionarily conserved sig-

naling 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 main-

tenance (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.

Results

The JAK/STAT pathway activity in theDrosophila midgut

In a search for additional signaling pathways involved in ISC regu-

lation, 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 b-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 �

STAT-GFP is a GFP reporter with 10 copies of STAT-binding sites

at the promoter, which serves as a reliable marker for STAT92E acti-

vation 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 pro-

duced 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 ISCmaintenance 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 Dlþ ISCs from total epithelial

cells was 9.8+0.96% (intestines examined, n ¼ 5) in upd4/þ

intestines, 5.6+0.38% (n ¼ 4) in upd4/ossiscG20 intestines and

7.0+0.54% (n ¼ 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 ¼ 5) in upd4/þ ,

4.0+0.60% (n ¼ 5) in upd4/ossiscG20 and 4.3+0.56% (n ¼ 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.

Canonical JAK/STAT signaling componentsare cell-autonomously required for ISCmaintenance

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 exam-

ining their maintenance and proliferation over time, as previously

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 3

described (Lin et al., 2008). The initially GFP-marked normal or

mutant ISCs were generated by heat shock-induced mitotic recom-

bination 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 differen-

tiated 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 homo-

zygous 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 mainten-

ance, 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

Figure 2 upd is necessary for ISC maintenance. Superficial sections through the epithelium of upd heterozygous (A) and trans-heterozygous (B)

intestines stained with anti-Dl antibody (red) and DAPI (blue). Scale bar, 10 mm. Graphs show quantitative analysis of ISC number (C) and

mitotic index (D) in control and mutant intestines. Error bars indicate SEM. n ¼ 4–7.

4 j Journal of Molecular Cell Biology Lin et al.

division rate. As a control, the wild-type ISCs had 30.5% (n ¼ 236)

labeled with BrdU at 1 week after clone induction. In contrast,

domeG0468, domeG0405, hopC111and hopM4 mutant ISCs had only

14.3% (n ¼ 28), 19.0% (n ¼ 21), 10.7% (n ¼ 28) and 20.0%

(n ¼ 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.

Figure 3 JAK/STAT signaling is essential for ISC maintenance and lineage differentiation. (A–G) Examples of typical GFP clones (green) of given

genotypes at 2 weeks after clone induction (anti-Dl, red, membrane; anti-Pros, red, nucleus). (H) A FLP-OUT GFP clone with forced domeDN

expression at 2 weeks after clone induction. Note that the GFP cells were negative for Dl expression (red), and showed diploid-like cell

sizes. (I) Time course analysis of wild-type and mutant ISC clones. The percentage of ISC clones at a given time point is calculated as the

number of clones carrying Dlþ cells in each midgut divided by the average number of Dlþ clones in each midgut at Day 4. Error bars indicate

SEM. n ¼ 10–20 (intestines examined for each genotype at each time point). In all images, DAPI staining was in blue. Scale bar, 10 mm.

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 5

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 regu-

late the maintenance and proliferation of ISCs.

JAK/STAT and Wingless signaling function inparallel for ISC maintenance andproliferation

Because Wg signaling has a similar role in regulating ISC mainten-

ance 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)/b-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 be-

havior 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 ¼ 24) for arm3 domeG0468 mutant

ISCs and 8.3% (n ¼ 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 indi-

cates that JAK/STAT and Wg signaling function cooperatively and in

parallel in regulating ISC maintenance and proliferation.

The JAK/STAT pathway is essential forintestinal lineage differentiation

We noticed that in dome and hop homozygous mutant ISC-derived

clones, polyploid EC or Prosþ 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 progeni-

tor 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

Dlþ ISCs. It also produced many Dl2 GFPþ small diploid cells

(Figure 4A and A0), 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 4B0). Because N is essential for EB differen-

tiation, 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 4C0), 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.

Therefore, JAK/STAT signaling may have a critical role in pro-

moting 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 particu-

larly critical for ee cell differentiation.

JAK signaling activation causes a limitedexpansion of the ISC population but adramatic 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

Table 1 JAK/STAT signaling is essential for ee cell and EC

differentiation.

% ISC clone with EC* % ISC clone with ee*

wt clone (2 weeks) 100.0% (106/106) 58.5% (62/106)

wt clone (3 weeks) 100.0% (125/125) 44.8% (56/125)

domeG0468 (2 weeks) 16.7% (5/30) 6.7% (2/30)

hopc111 (2 weeks) 24.1% (7/29) 6.9% (2/29)

domeDN (2 weeks) 75.0% (21/28) 7.1% (2/28)

stat06346 (3 weeks) 86.6% (58/67) 9.0% (7/67)

*The percentage of ISC clones with EC/ee was calculated by the number of EC/

ee-containing ISC clones examined at a given time point divided by the total

number of ISC clones examined. EC, enterocyte; ee, enteroendocrine cell.

6 j Journal of Molecular Cell Biology Lin et al.

ISCs are scattered along the basement membrane of the epithe-

lia, 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 over-

expression also produced a weak phenotype similar to that

caused by Arm overexpression (Figure 5B and C) and the

Figure 4 JAK/STAT signaling regulates EB differentiation. (A and A0) Dome RNAi (genotype: esg-Gal4,UAS-GFP/UAS-dome-RNAi; tub-Gal80ts/ þ )

caused accumulation of EB-like cells (Dl2, GFPþ). GFP (green), anti-Dl (red) and DAPI (blue). (B and B0) A domeG0468 mutant clone marked by GFP

(green) stained with antibodies against Pdm-1 (red). The mutant cells (arrow) were negative for Pdm-1 expression. (C and C0) hopC111 mutant

clones marked by GFP (green) stained with Su(H)m8-lacZ (red). The mutant cells (arrow) were positive for LacZ expression. Scale bar, 10 mm.

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 7

originally observed stronger phenotype was possibly contribu-

ted by certain genetic background. Forced expression of upd

or hop also caused weak expansion of ISC-like cells (Dlþ,

GFPþ) and EB-like cells (Dl2, GFPþ) (Figure 5D–E). Unlike Wg

signaling activation, however, overexpression of upd or hop

also led to a dramatic increase of ee-like cells (Prosþ, GFP2)

in the epithelium (Figure 5D–E0). Many of these Prosþ 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 ¼ 5), respectively, in

upd overexpressed intestines and 18.4+3.07% and 52.7+7.24% (n ¼ 5) in hop overexpressed intestines, compared

with 6.9+0.47% and 61.7+1.38% (n ¼ 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.

Figure 5 Effects of JAK/STAT or Wg signaling overactivation on ISC lineages. (A) Control. Flies (genotype: esg-Gal4,UAS-GFP/ þ ; tubGal80ts/ þ )

grow at 298C for 2 weeks. (B–E0) Experimental. Flies with various transgene expression grow at 298C for 2 weeks. (B and B0) Arm activation (gen-

otype: esg-Gal4,UAS-GFP/UAS-armDN; tubGal80ts/ þ ) caused weak expansion of ISC-like cells (Dlþ, GFPþ) and EB-like cells (Dl2, GFPþ) without

complete blockage in terminal differentiation, as ECs and ee cells were still produced. (C) wg overexpression caused similar phenotypes as Arm

activation. (D–E0) hop overexpression (D and D0) as well as upd overexpression (E and E0) caused a weak expansion of ISC-like cells (Dlþ, GFPþ,

arrows) and EB-like cells (Dl2, GFPþ) and a strong expansion of ee cell population (Prosþ, GFP2). (F) A plot showing the percentages of EC and ee

cell population in wild-type and upd or hop overexpressed intestines. Error bars indicate SEM. n ¼ 5–8. GFP (green), anti-Dl (red, membrane),

anti-Pros antibody (red, nucleus) and DAPI (blue). Scale bar, 10 mm.

8 j Journal of Molecular Cell Biology Lin et al.

Co-activation of JAK and Wg signalingsynergistically 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-armDN or UAS-upd and UAS-armDN. Simultaneous acti-

vation of both pathways caused dramatic enhancement of

ISC-like cell (Dl2, GFPþ) accumulation than by activating either

pathway alone (Figure 6). Co-expression of UAS-upd and

UAS-armDN also frequently produced spherical-shaped tumor-

like cell masses that were filled with ISC-like cells (Dlþ, GFPþ)

and progenitor cells (Dl2, GFPþ) (Figure 6B and B0).

Consequently, the intestinal tract frequently displayed disorga-

nized morphology at these regions (Figure 6B and B0). 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 conse-

quence of both enhanced ISC self-renewal and blockage in differ-

entiation. 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).

Antagonistic activities of JAK and Notchcontrol binary fate choice of intestinalprogenitors

Previous studies suggest that N functions downstream of Wg sig-

naling 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), Dlþ 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 sig-

naling is required for N mutation-induced ee cell-like tumor for-

mation, 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 differen-

tiation, we propose that Nhigh and JAKlow allow EB differentiation

into EC, whereas Nlow and JAKhigh allow EB differentiation into ee

Figure 6 JAK/STAT and Wg signaling co-activation synergistically promotes ISC self-renewal. Co-activation of Wg and JAK/STAT signaling by

expressing UAS-armDN and UAS-hop (A) or UAS-armDN and UAS-upd (B) causes strong expansion of ISC-like (Dlþ, GFPþ) cell populations.

UAS-armDN and UAS-upd expression also produced large spherical cell masses that led to abnormal intestinal morphology (the dashed line

in B). (A0 and B0) The red channels of images A and B, respectively. GFP: green; anti-Dl: red (membrane); anti-Pros: red (nucleus); DAPI:

blue. Scale bar, 10 mm.

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 9

cell, and antagonistic activities of N and STAT signaling control

the binary decision between EC and ee cell fate (Figure 7D).

DiscussionIn this study, we identified two novel and separate roles of JAK/

STAT signaling in ISC lineages in the Drosophila midgut. It func-

tions 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 Dlþ esgþ 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

Figure 7 Genetic relationships between JAK/STAT and N signaling in ISC self-renewal. (A) N264-40 mutant clones at 2 weeks after clone induction

displayed either ISC-like (Dlþ, GFPþ) or ee cell-like tumors (Prosþ, GFPþ, dashed line). (B) N264-40 domeG0468 double-mutant clones at 2 weeks

after clone induction exhibited similar ISC-like tumor phenotypes (Dlþ, GFPþ), but ee cell-like tumor was rarely observed. In some cases, only a

small cluster of ee-like cells was observed (dashed line). (C) Similarly, a stat92E06346 mutant clone with UAS-Notch-RNAi expression also pro-

duced ISC-like tumors, but ee cell-like tumor was rarely observed. GFP: green; anti-Dl: red (membrane); anti-Pros: red (nucleus); DAPI: blue.

Scale bar, 10 mm. (D) A proposed model for controlling ISC self-renewal and differentiation by signaling pathways. Upd, Wg and possibly other

secreted signals from the muscular niche function together to control ISC self-renewal by antagonizing N activity, and differential activities of N

and JAK/STAT signaling determine the binary cell fate decision between EC and ee cell from EB. BM, basement membrane; ISC, intestinal stem

cell; EB, enteroblast; EC, enterocyte; ee, enteroendocrine cell; N, Notch.

10 j Journal of Molecular Cell Biology Lin et al.

adenomatous polyposis coli (Apc) or Axin promotes ISC prolifer-

ation but does not seem to affect ISC self-renewal (Lee et al.,

2009). However, co-activation of Wg and JAK signaling dramati-

cally increases Dlþ esgþ 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 reg-

ulating 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 absol-

utely 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 acti-

vated 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), indi-

cating that under stress conditions, niche signals could be regu-

lated 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 differ-

entiation toward the ee cell fate, whereas a low level of JAK acti-

vation 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 developmen-

tal 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 conse-

quently 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 interest-

ing 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 differen-

tiation. 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 facili-

tate N activation in EBs to promote their differentiation into ECs

rather than ee cells (Maeda et al., 2008). We suggest that a sig-

naling 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 specifi-

cally 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 Olfm4þ cells within the crypt

region (van der Flier et al., 2009). In addition, Wnt signaling acti-

vation 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 obser-

vations 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 gov-

erning 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.

Materials and methods

Fly strains

Flies were maintained at 258C 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 � STAT92E-GFP; dome-MESO(dome-lacZ); UAS-dome4CYT

(UAS-domeDN); UAS-wingless; UAS-upd(PK9); UAS-hop(3W);

UAS-arm4N (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/þ , tub-gal80 FRT19A/FRT19A;

Act-Gal4, UASGFP/þ ; hsflp122/þ , tub-gal80 FRT19A/upd4

FRT19A; Act-Gal4, UAS-GFP/þ ; hsflp122/þ , tub-gal80 FRT19A/

domeG0468 FRT19A; Act-Gal4, UAS-GFP/þ ; hsflp122/þ ,

tub-gal80 FRT19A/hopC111 FRT19A; Act-Gal4, UAS-GFP/þ ;

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 11

hsflp122/þ ; Act-Gal4, UAS-GFP/þ ; Tub-Gal80 FRT82B/

stat92E06346 FRT82B; hsflp122/þ , tub-gal80 FRT19A/

arm3domeG0468 FRT19A; Act-Gal4, UAS-GFP/þ ; hsflp122/þ ,

tub-gal80 FRT19A/arm3hopC111 FRT19A; Act-Gal4, UAS-GFP/þ ;

hsflp122/þ , tub-gal80 FRT19A/N264-40domeG0468 FRT19A;

Act-Gal4, UAS-GFP/þ ; hsflp122/UAS-Notch dsRNA; Act-Gal4,

UAS-GFP/þ ; Tub-Gal80 FRT82B/stat6346 FRT82B. Clones were

induced by 1 h heat shock treatments of 3–5-day-old females in

a 378C running water bath. To overexpress domeDN using the

FLP-OUT cassette, the following adults of genotype were gener-

ated: hsflp122/þ ; Act5C . y . Gal4, UAS-GFP/UAS-dome4N.

Flies were heat-shocked at 378C 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 188C, and newly enclosed flies of appro-

priate genotypes were shifted to 298C for 2 weeks before dissec-

tion. 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 dis-

sected 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 48C overnight. Secondary antibodies were incubated for

either 2 h at room temperature or overnight at 48C. 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 Nassel, 1:3000); rabbit anti-BrdU anti-

body (1:300); rabbit anti-phospho-Histone H3 antibody (Upstate,

1:1000); rabbit polyclonal anti-b-gal antibodies (Cappel, 1:6000);

Alexa-568-conjugated goat anti-mouse/rabbit and

Alexa-488-conjugated goat anti-rabbit/mouse secondary anti-

bodies (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 mg/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 378C. The reac-

tion 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 pre-

viously 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.

AcknowledgementsWe 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 Nassel, 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.

FundingThis work was supported by an ‘863’ grant (2007AA02Z1A2 to

R.X.) from the Chinese Ministry of Science and Technology.

ReferencesAgaisse, H., Petersen, U.M., Boutros, M., Mathey-Prevot, B., and Perrimon, N.

(2003). Signaling role of hemocytes in Drosophila JAK/STAT-dependent

response to septic injury. Dev. Cell 5, 441–450.

Amcheslavsky, A., Jiang, J., and Ip, Y.T. (2009). Tissue damage-induced intesti-

nal stem cell division in Drosophila. Cell Stem Cell 4, 49–61.

Arbouzova, N.I., and Zeidler, M.P. (2006). JAK/STAT signalling in Drosophila:

insights into conserved regulatory and cellular functions. Development

133, 2605–2616.

Assa-Kunik, E., Torres, I.L., Schejter, E.D., Johnston, D.S., and Shilo, B.Z.

(2007). Drosophila follicle cells are patterned by multiple levels of Notch sig-

naling and antagonism between the Notch and JAK/STAT pathways.

Development 134, 1161–1169.

Bach, E.A., Ekas, L.A., Ayala-Camargo, A., Flaherty, M.S., Lee, H., Perrimon, N.,

and Baeg, G.H. (2007). GFP reporters detect the activation of the Drosophila

JAK/STAT pathway in vivo. Gene Expr. Patterns 7, 323–331.

12 j Journal of Molecular Cell Biology Lin et al.

Baltgalvis, K.A., Berger, F.G., Pena, M.M., Davis, J.M., Muga, S.J., and Carson,

J.A. (2008). Interleukin-6 and cachexia in ApcMin/þ mice. Am. J. Physiol.

Regul. Integr. Comp. Physiol. 294, R393–R401.

Barker, N., Ridgway, R.A., van Es, J.H., van de Wetering, M., Begthel, H., van

den Born, M., Danenberg, E., Clarke, A.R., Sansom, O.J., and Clevers, H.

(2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature

457, 608–611.

Binari, R., and Perrimon, N. (1994). Stripe-specific regulation of pair-rule genes

by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes

Dev. 8, 300–312.

Bollrath, J., Phesse, T.J., von Burstin, V.A., Putoczki, T., Bennecke, M., Bateman,

T., Nebelsiek, T., Lundgren-May, T., Canli, O., Schwitalla, S., et al. (2009).

gp130-mediated Stat3 activation in enterocytes regulates cell survival and

cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell

15, 91–102.

Brand, A.H., and Perrimon, N. (1993). Targeted gene expression as a means of

altering cell fates and generating dominant phenotypes. Development 118,

401–415.

Brown, S., Hu, N., and Hombria, J.C. (2001). Identification of the first invert-

ebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Curr.

Biol. 11, 1700–1705.

Brown, S., Hu, N., and Hombria, J.C. (2003). Novel level of signalling control in

the JAK/STAT pathway revealed by in situ visualisation of protein–protein

interaction during Drosophila development. Development 130, 3077–3084.

Buchon, N., Broderick, N.A., Poidevin, M., Pradervand, S., and Lemaitre, B.

(2009). Drosophila intestinal response to bacterial infection: activation of

host defense and stem cell proliferation. Cell Host Microbe 5, 200–211.

Casali, A., and Batlle, E. (2009). Intestinal stem cells in mammals and

Drosophila. Cell Stem Cell 4, 124–127.

Chen, H.W., Chen, X., Oh, S.W., Marinissen, M.J., Gutkind, J.S., and Hou, S.X.

(2002). mom identifies a receptor for the Drosophila JAK/STAT signal trans-

duction pathway and encodes a protein distantly related to the mammalian

cytokine receptor family. Genes Dev. 16, 388–398.

Cronin, S.J., Nehme, N.T., Limmer, S., Liegeois, S., Pospisilik, J.A., Schramek,

D., Leibbrandt, A., Simoes, R.D., Gruber, S., Puc, U., et al. (2009).

Genome-wide RNAi screen identifies genes involved in intestinal pathogenic

bacterial infection. Science 325, 340–343.

Flaherty, M.S., Zavadil, J., Ekas, L.A., and Bach, E.A. (2009). Genome-wide

expression profiling in the Drosophila eye reveals unexpected repression

of notch signaling by the JAK/STAT pathway. Dev. Dyn. 238, 2235–2253.

Furriols, M., and Bray, S. (2001). A model Notch response element detects

suppressor of hairless-dependent molecular switch. Curr. Biol. 11, 60–64.

Gilbert, M.M., Weaver, B.K., Gergen, J.P., and Reich, N.C. (2005). A novel func-

tional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed

by an in vivo reporter of pathway activation. Mech. Dev. 122, 939–948.

Gregory, L., Came, P.J., and Brown, S. (2008). Stem cell regulation by JAK/STAT

signaling in Drosophila. Semin. Cell Dev. Biol. 19, 407–413.

Grivennikov, S., Karin, E., Terzic, J., Mucida, D., Yu, G.Y., Vallabhapurapu, S.,

Scheller, J., Rose-John, S., Cheroutre, H., Eckmann, L., et al. (2009). IL-6

and Stat3 are required for survival of intestinal epithelial cells and develop-

ment of colitis-associated cancer. Cancer Cell 15, 103–113.

Harrison, D.A., McCoon, P.E., Binari, R., Gilman, M., and Perrimon, N. (1998).

Drosophila unpaired encodes a secreted protein that activates the JAK sig-

naling pathway. Genes Dev. 12, 3252–3263.

Hombria, J.C., Brown, S., Hader, S., and Zeidler, M.P. (2005). Characterisation

of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 288, 420–433.

Hou, X.S., Melnick, M.B., and Perrimon, N. (1996). Marelle acts downstream of

the Drosophila HOP/JAK kinase and encodes a protein similar to the mam-

malian STATs. Cell 84, 411–419.

Jiang, H., Patel, P.H., Kohlmaier, A., Grenley, M.O., McEwen, D.G., and Edgar,

B.A. (2009). Cytokine/Jak/Stat signaling mediates regeneration and homeo-

stasis in the Drosophila midgut. Cell 137, 1343–1355.

Lee, T., Winter, C., Marticke, S.S., Lee, A., and Luo, L. (2000). Essential roles of

Drosophila RhoA in the regulation of neuroblast proliferation and dendritic

but not axonal morphogenesis. Neuron 25, 307–316.

Lee, W.C., Beebe, K., Sudmeier, L., and Micchelli, C.A. (2009). Adenomatous

polyposis coli regulates Drosophila intestinal stem cell proliferation.

Development 136, 2255–2264.

Lin, G., and Xi, R. (2008). Intestinal stem cell, muscular niche and Wingless sig-

naling. Fly (Austin) 2, 310–312.

Lin, G., Xu, N., and Xi, R. (2008). Paracrine Wingless signalling controls self-

renewal of Drosophila intestinal stem cells. Nature 455, 1119–1123.

Logan, C.Y., and Nusse, R. (2004). The Wnt signaling pathway in development

and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810.

Maeda, K., Takemura, M., Umemori, M., and Adachi-Yamada, T. (2008).

E-cadherin prolongs the moment for interaction between intestinal stem

cell and its progenitor cell to ensure Notch signaling in adult Drosophila

midgut. Genes Cells 13, 1219–1227.

McGregor, J.R., Xi, R., and Harrison, D.A. (2002). JAK signaling is somatically

required for follicle cell differentiation in Drosophila. Development 129,

705–717.

McGuire, S.E., Le, P.T., Osborn, A.J., Matsumoto, K., and Davis, R.L. (2003).

Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302,

1765–1768.

Micchelli, C.A., and Perrimon, N. (2006). Evidence that stem cells reside in the

adult Drosophila midgut epithelium. Nature 439, 475–479.

Morrison, S.J., and Spradling, A.C. (2008). Stem cells and niches: mechanisms

that promote stem cell maintenance throughout life. Cell 132,

598–611.

Ohlstein, B., and Spradling, A. (2006). The adult Drosophila posterior midgut is

maintained by pluripotent stem cells. Nature 439, 470–474.

Ohlstein, B., and Spradling, A. (2007). Multipotent Drosophila intestinal stem

cells specify daughter cell fates by differential notch signaling. Science 315,

988–992.

Rawlings, J.S., Rosler, K.M., and Harrison, D.A. (2004). The JAK/STAT signaling

pathway. J. Cell Sci. 117, 1281–1283.

Struhl, G., and Basler, K. (1993). Organizing activity of wingless protein in

Drosophila. Cell 72, 527–540.

Takashima, S., Mkrtchyan, M., Younossi-Hartenstein, A., Merriam, J.R., and

Hartenstein, V. (2008). The behaviour of Drosophila adult hindgut stem

cells is controlled by Wnt and Hh signalling. Nature 454, 651–655.

van den Flier, L.G., and Clevers, H. (2009). Stem cells, self-renewal, and differ-

entiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260.

van den Flier, L.G., van Gijn, M.E., Hatzis, P., Kujala, P., Haegebarth, A., Stange,

D.E., Begthel, H., van den Born, M., Guryev, V., Oving, I., et al. (2009).

Transcription factor achaete scute-like 2 controls intestinal stem cell fate.

Cell 136, 903–912.

Yan, R., Small, S., Desplan, C., Dearolf, C.R., and Darnell, J.E., Jr. (1996).

Identification of a Stat gene that functions in Drosophila development.

Cell 84, 421–430.

Zecca, M., Basler, K., and Struhl, G. (1996). Direct and long-range action of a

wingless morphogen gradient. Cell 87, 833–844.

JAK/STAT signaling in Drosophila ISCs Journal of Molecular Cell Biology j 13