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RESEARCH ARTICLE

Integrins regulate epithelial cell differentiation by modulatingNotch activity

M. Jesus Gomez-Lamarca1, Laura Cobreros-Reguera1, Beatriz Ibanez-Jimenez1, Isabel M. Palacios2 andMarıa D. Martın-Bermudo1,*

ABSTRACT

Coordinating exit from the cell cycle with differentiation is crucial for

proper development and tissue homeostasis. Failure to do so can

lead to aberrant organogenesis and tumorigenesis. However, little is

known about the developmental signals that regulate the switch

from cell cycle exit to differentiation. Signals downstream of two key

developmental pathways, Notch and Salvador–Warts–Hippo

(SWH), and signals downstream of myosin activity regulate this

switch during the development of the follicle cell epithelium of the

Drosophila ovary. Here, we have identified a fourth player, the

integrin signaling pathway. Elimination of integrin function blocks the

mitosis-to-endocycle switch and differentiation in posterior follicle

cells (PFCs), by regulation of the cyclin-dependent kinase inhibitor

(CKI) dacapo. In addition, integrin-mutant PFCs show defective

Notch signaling and endocytosis. Furthermore, integrins act in

PFCs by modulating the activity of the Notch pathway, as reducing

the amount of Hairless, the major antagonist of Notch, or

misexpressing Notch intracellular domain rescues the cell cycle

and differentiation defects. Taken together, our findings reveal a

direct involvement of integrin signaling on the spatial and temporal

regulation of epithelial cell differentiation during development.

KEY WORDS: Integrins, Proliferation, Differentiation

INTRODUCTIONThe processes of cell proliferation and differentiation have been

studied independently, but the cell cycle is intimately entwined

with differentiation, as the latter is usually accompanied by

irreversible cell cycle exit. However, in some cell types, a

modified cell cycle occurs during differentiation, in which the

DNA is replicated without concomitant cell division, resulting in

an increase in nuclear DNA content. This process, called

endoreplication, occurs in many cell types, including plant,

invertebrate and vertebrate epidermis and in platelet-producing

megakaryocytes and placental trophoblast cells in mammals.

Furthermore, recent work indicates that progression through the

endoreplication cycle is an important aspect of cell fate

acquisition and cell differentiation in Arabidopsis (Bramsiepe

et al., 2010). However, although considerable progress has been

made in understanding how the core cell cycle machinery is

modified to convert a mitotic cycle into an endocycle, little is

known about the developmental signals that couple the mitosis-

to-endocycle switch with differentiation.

The follicular epithelium of the Drosophila ovary constitutes an

excellent model system for the in vivo study of the molecular and

cellular mechanisms regulating the mitotic to endocycle switch and

its association with cell differentiation. The Drosophila ovary is

composed of ,15 ovarioles, each containing a germarium at their

proximal end and progressively older egg chambers at their distal

end. Each egg chamber is composed of 15 nurse cells and one

oocyte surrounded by a single layer of follicle cells, which

constitute the follicular epithelium (King, 1970). Follicle cells

originate from the asymmetric division of the follicle stem cells

(FCSs), which are located in the germarium (Margolis and

Spradling, 1995). At the time that the egg chamber buds from

the germarium, ,80 follicle cells surround the germline cyst

(King, 1970). Follicle cells continue to divide mitotically until

stage 6 (S6). At this point, they exit the mitotic cycle and switch to

an endocycle (Calvi et al., 1998). Between S7 and S10, follicle

cells undergo three rounds of endoreplication, become polyploid

and increase their size. At S10B, these cells undergo their third cell

cycle regimen, gene amplification, during which four regions

containing genes encoding chorion proteins are locally amplified

(Calvi et al., 1998). These switches in cell cycle are coupled with

changes in cell growth and differentiation.

A major pathway controlling cell proliferation and

differentiation in follicle cells is the Notch pathway. The Notch

pathway is evolutionary conserved and operates iteratively in an

enormous diversity of developmental and physiological processes

(Andersson et al., 2011; Bray, 2006). This indicates that, despite

its simple molecular design, the Notch pathway must be able to

elicit a large variety of appropriate responses in many spatially

and temporally distinct cellular contexts. This versatility in

signaling output is, in part, achieved by modulation of the

pathway at different levels in the signal transduction cascade,

such as post-translational modifications and trafficking of both

the receptor itself and its ligands (reviewed in Bray, 2006). Notch

activity is also highly sensitive to chromatin modification and

histone rearrangements (reviewed in Andersson et al., 2011;

Bray, 2006). Finally, a number of reports have indicated that

crosstalk with other signaling pathways also influences the Notch

signaling output, adding additional levels of regulation (reviewed

in Guruharsha et al., 2012). In the context of this work, the Notch

pathway has been shown to regulate the mitotic-to-endocycle

switch, as well as the transition from undifferentiated to mature

follicle cells (reviewed in Klusza and Deng, 2011). At around S6,

increased Delta (Dl) expression in the germ line activates Notch

in follicle cells, promoting the mitotic-to-endocycle switch (Deng

et al., 2001; Lopez-Schier and St Johnston, 2001). Loss of

1Centro Andaluz de Biologıa del Desarrollo CSIC-University Pablo de Olavide,Sevilla 41013, Spain. 2The Department of Zoology, University of Cambridge,Downing Street, Cambridge CB2 3EJ, UK.

*Author for correspondence (mdmarber@upo.es)

Received 17 March 2014; Accepted 18 August 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4667–4678 doi:10.1242/jcs.153122

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function of Notch in follicle cells, or Dl in the germ line, leadsto prolonged mitosis at the expense of endocycles and to a block

in cell differentiation. Notch signaling negatively regulatesthe Drosophila Cdc25 phosphatase String (Stg). Because Stgactivates the CycA–Cdk1 and CycB–Cdk1 complexes and aninhibitor of CycE–Cdk2 complexes – the p27Cip/Kip cyclin-

dependent kinase inhibitor (CKI) protein Dacapo (Dap) – Notchsignaling allows CycE to oscillate, driving cells into theendocycle. In addition, Notch positively regulates Fizzy-related

(Fzr), an adaptor for the APC/C E3 ligase, which allows thedegradation of the mitotic cyclins, thereby ensuring completetransition to endocycle (Schaeffer et al., 2004; Shcherbata et al.,

2004). One pathway that has been shown to modulate Notchactivity in this context is the Salvador–Warts–Hippo (SWH)pathway (Yu et al., 2008). SWH-mutant follicle cells continue

proliferating, fail to enter into the endocycle and remainundifferentiated (Meignin et al., 2007; Polesello and Tapon,2007; Yu et al., 2008). Furthermore, the SWH pathway has beenproposed to promote Notch signaling in follicle cells by

regulating Notch endocytic trafficking (Meignin et al., 2007;Polesello and Tapon, 2007; Yu et al., 2008). The Notch pathwaycan also be modulated by myosin activity in follicle cells. As it is

the case for SWH, elimination of PP1b, a phosphatase thatdephosphorylates and inactivates the non-muscle myosin II lightchain, also results in defective Notch signaling, owing to defects

in the endosomal trafficking of the receptor (Sun et al., 2011).Interestingly, myosin activity does not affect the SWH pathway.These results highlight the complexity of the genetic circuitry that

affects Notch signaling during follicle cell differentiation.In this work, we have identified a fourth pathway regulating the

cell-proliferation-to-differentiation switch in follicle cells –signaling downstream of integrins. Integrins are ab heterodimeric

cell-surface receptors that connect the extracellular matrix (ECM)to the cytoskeleton (Hynes, 2002). Although in vertebrates there areat least eight b subunits and 18 a subunits, in Drosophila, there are

only two b subunits, bPS and bn, and five a subunits, aPS1 to aPS5.We have shown previously that elimination of myospheroid (mys),the gene encoding the bPS subunit (the only b subunit present in the

ovary), from follicle cells results in multilayering and polaritydefects at both poles of the egg chamber (Fernandez-Minan et al.,2008). Here, we report a new role for integrins in follicle cells – thecontrol of the coupling of mitosis-to-endocycle switch with

differentiation. We show that elimination of integrin functionresults in aberrant PFC endoreplication and differentiation.Furthermore, we show that integrins regulate proper exit from the

cell cycle and cell differentiation by regulating the expression of theCDK inhibitor dacapo. Our results also demonstrate that integrinsact in follicle cells by modulating the activity of the Notch pathway

through the regulation of its intracellular trafficking and/orprocessing. In summary, we believe that our work reveals thatcell–ECM interactions mediated by integrins contribute to the

temporal coupling of cell-cycle arrest with differentiation duringdevelopment.

RESULTSIntegrins regulate follicle cell differentiationWe have previously reported that mosaic egg chamberscontaining mys mutant follicle cell clones very often grow

extra layers at both poles of the egg chamber (Fernandez-Minanet al., 2007). In addition, we have shown that mys-mutant folliclecells located at the posterior pole, hereafter referred to as mys

PFCs, show an aberrant distribution of polarity markers

(Fernandez-Minan et al., 2008). Interestingly, this polaritydefect is only observed in mys PFCs located in the ectopic

layers, whereas mys PFCs in contact with the germline are correctlypolarized (Fernandez-Minan et al., 2008). Recently, we noticedthat the nuclear diameter of mys PFCs (as identified by the absenceof GFP) was significantly smaller than that of their wild-type

counterparts (Fig. 1A,B). As was the case for the polarity defects,the nuclear size phenotype was only observed in mutant PFCs thathave lost contact with the germline (Fig. 1A, white arrow),

whereas mys PFCs in contact with the germline show no phenotype(Fig. 1, white arrowhead). Small nuclear size is a potentialindication of a failure to differentiate and to transit into the

endocycle. Thus, we checked whether integrins were required forproper follicle cell differentiation and mitotis-to-endocycle switch.In order to do this, we generated follicle cell clones for two

unrelated null alleles mys11 and mys10, and we analyzed theexpression of markers normally found in undifferentiated cells[FasIII and Eyes absent (Eya)] or in differentiated PFCs [pointed-

lacZ (pnt-lacZ) (Bai and Montell, 2002; Gonzalez-Reyes and St

Johnston, 1998; Morimoto et al., 1996; Snow et al., 1989)]. Bothalleles gave rise to indistinguishable phenotypes; however, in thetext and in the figures we refer only to results obtained with mys11.

In wild-type egg chambers, FasIII and Eya are detected in allfollicle cells up to S6 (insets in Fig. 1C,D, respectively). From S6onwards, they are expressed only in polar cells (FasIII) or border

cells and stretched cells (Eya) (Fig. 1C,D). By contrast, pnt-lacZ isspecifically expressed in wild-type PFCs from S6 onwards(Fig. 1C,D). However, in contrast to the wild type, we found that

in S6 and older egg chambers, most mys PFCs express FasIII andEya (Fig. 1E, 83% n537; Fig. 1F, 80%, n542, respectively) andfail to activate pnt-lacZ (Fig. 1E,F, 100%, n531). Again, theeffects on FasIII, Eya and pnt-lacZ expression were only observed

in mys PFCs located in the ectopic layers (Fig. 1E,F, arrows).Finally, we found that the RNA-binding protein Staufen (Stau),which localises to the posterior of the oocyte during S9 (St

Johnston et al., 1991; supplementary material Fig. S1A), wasproperly localised in mosaic egg chambers containing mys PFCs(supplementary material Fig. S1B, arrowhead), most likely owing

to the normal differentiation of mys PFCs in contact with the germline. Taken together, these results strongly suggest that integrinsare required for proper PFC maturation when contact with thegermline is lost.

Integrins are required in follicle cells for the mitosis-to-endocycle switch and for gene amplificationDifferentiation and withdrawal from the mitotic cycle are coupledin follicle cells. However, whether initiation of the differentiationprogram in follicle cells depends on exit from cell cycle has not

yet been formally proven. Here, we decided to directly addressthis by blocking exit from the cell cycle through the simultaneousoverexpression of E2F, Dp (DP transcription factor), CycE and Stg.

This combination is sufficient to abrogate cell cycle exit inDrosophila eye and wing disc cells, where cell cycle arrest is notrequired for terminal differentiation (Buttitta et al., 2007). Here, wefound that overexpression of those four genes in follicle cells was

also sufficient to bypass cell cycle exit, as visualized by ectopicphosphorylated histone 3 (PH3) staining after S6 (supplementarymaterial Fig. S2B). However, in contrast to the wing and eye,

differentiation in follicle cells was blocked, as revealed by themaintenance of high levels of FasIII expression after S6(supplementary material Fig. S2B). These results strongly suggest

that follicle cells need to exit the mitotic cycle to differentiate.

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We show here that mys PFCs display differentiation defects.This could reflect a failure to exit the mitotic cycle or a problem

with differentiation. To test between these possibilities, weanalyzed the expression of the cell cycle regulator Cyclin B(CycB). CycB is normally expressed in mitotic follicle cells from

S4 to S6, when it is downregulated (Lehman et al., 1999; inset inFig. 2A). However, we found that mys PFCs located in ectopiclayers showed prolonged expression of CycB after S6 (Fig. 2B,

62%, n534, arrow). We next analyzed the expression of theBroad-complex (BR-C) gene, an early ecdysone-response genethat is essential for endoreplication and gene amplification. Inwild-type egg chambers, the expression of the BR-C protein is

activated by ecdysone in all follicle cells from S6 onwards(Tzolovsky et al., 1999; Fig. 2C), where it drives geneamplification, as visualised by the incorporation of BrdU in

four discrete spots that represent amplification of the choriongenes in S10B follicle cells (Fig. 2E). However, we did not detectBR-C expression in mys PFCs located in ectopic layers after S6

(Fig. 2D, 100%, n539, arrow). As expected, these mys PFCs alsofail to undergo gene amplification, as shown by BrdUincorporation (Fig. 2F, 75%, n538, arrow). Thus, instead of the

foci-like pattern of BrdU incorporation typical of follicle cellsundergoing gene amplification (Fig. 2E), some S10B mys PFCsshow a pattern characteristic of cells in mitosis, where BrdUlabels the whole nucleus (Fig. 2F, arrow). CycB and BR-C

expression patterns were not affected in mys PFCs in contact withthe germline (arrowheads in Fig. 2B,D).

Taken together, our results show that mys PFCs located inectopic layers fail to undergo proper mitotic-to-endocycle switch

and cell differentiation. As mentioned in the Introduction, a keypathway regulating cell proliferation and differentiation in folliclecells is the Notch pathway. Thus, we next decided to analyze

whether elimination of integrins could affect the Notch pathway.

Notch signaling is disrupted in mys PFCsTo examine whether Notch signaling was affected in mys PFCs,we analyzed the expression of Cut, a homeobox protein that isdownregulated by Notch signaling in follicle cells after S6 (Sunand Deng, 2005; Fig. 3A), and that of Hindsight (Hnt, also known

as Pebbled), a zinc-finger protein that is induced by Notch afterS6 (Sun and Deng, 2007; Fig. 3C). We found that, in contrast tothe wild-type PFCs, mys PFCs that had lost contact with the

germline showed continued expression of Cut (Fig. 3B, n527,arrow) and failed to activate Hnt (Fig. 3D, n529, arrow) in 82%and 80% of S6 and older egg chambers analyzed, respectively.

These results suggest that Notch signaling is affected in theabsence of integrin function. To further confirm this, wemeasured Notch activity using the Gbe-Su(H)-lacZ reporter,

which yields high levels of expression in cells where Notch isactive (Furriols and Bray, 2001; Fig. 3E). Normally, the activityof this reporter is increased in follicle cells upon Notch activationduring S7–S10A, although its expression is not uniform along the

follicular epithelium, being highest in PFCs (Fig. 3E). We foundthat the expression of this reporter was significantly reduced in

Fig. 1. Integrins regulate PFC differentiation. (A) An S9 mosaic egg chamber carrying mys mutant clones (GFP2) was stained for anti-GFP and the DNAmarker TO-PRO-3 (blue). Although mys PFCs in contact with the germline (GFP2, arrowhead) have normal nuclear size, the nuclear size of those locatedin ectopic layers (GFP2, arrow) is smaller than that of surrounding wild-type (wt) cells (GFP+). (B) Quantification of the nuclear size. Data show the mean6s.d.(C–F) S9–S10 wild-type and mosaic egg chambers carrying mys mutant clones expressing the posterior cell fate marker pnt-lacZ, and stained withanti-GFP (green), anti-b-galactosidase (C–F; blue), anti-FasIII (C,E; red) and anti-Eya (D,F; red). In S9–S10 wild-type egg chambers, FasIII (C) and Eya (D) arerestricted to polar cells (PCs; FasIII) or to border cells (BCs) and stretched cells (SCs; Eya), whereas pnt-lacZ is specifically expressed in PFCs from S6onwards (C,D). However, in mys PFCs located in ectopic layers (GFP2, arrow), FasIII (E) and Eya (F) are maintained, whereas pnt-lacZ is inhibited (E,F). mys

PFCs in contact with the germline (GFP2, arrowhead in E,F) behave as wild-type follicle cells. Upper-left panels in C and D show the expression of FasIII andEya in S4 egg chambers, respectively. Anterior is to the left in all figures. (A9–F9) Magnifications of the white boxes in A–F, respectively.

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mys PFCs located in ectopic layers in 75% of the egg chambersanalyzed (Fig. 3F, n528, arrow). These results show that Notchsignaling is indeed defective in mys PFCs that have lost contact

with the germline.

Integrins are required to strengthen Notch signalingAn intriguing aspect of the mys phenotype is the fact that the defectsin Notch signaling are only observed in mutant PFCs located inectopic layers, whereas those mys mutant cells in contact with the

germline develop normally. In this scenario, because the ligand forthe Notch receptor in the ovary, Dl, is produced in the germline(Deng et al., 2001), the mutant cells adjacent to the germline couldreceive a normal signal, activating the Notch pathway properly. By

contrast, those mutant cells in the ectopic layers, placed away fromthe source of ligand, would receive much less ligand input andwould consequently develop Notch-mutant phenotypes. Therefore,

the Notch-like defects could just be a secondary consequence of themultilayering phenotype. Alternatively, integrins might themselvesbe required to strengthen Notch signaling. This hypothetical

integrin modulation of Notch signaling would become morecrucial in a sensitized background, such as cells located in theectopic layers. To test between these two possibilities, we

performed four sets of experiments. First, we tested whetherincreasing Notch signaling in mys PFCs could rescue the phenotype.In order to do this, we removed one copy of Hairless (H), the majorantagonist of Notch signaling in Drosophila (reviewed in Maier,

2006), in mys mosaic egg chambers. Removing one copy of H in awild-type background did not affect follicle cell differentiation

(Fig. 4A). In addition, halving the dose of H in a mys background didnot have any effect on the onset or formation of the multilayering(Fig. 4C). However, interestingly, we found that it rescued the

differentiation (Fig. 4C,G, n532) and nuclear size defects (arrow inFig. 4C9,H, n528) of mys PFCs, as now only 23% of mosaic eggchambers containing mys PFCs and heterozygous for H (mys; H/+)

showed FasIII expression after S6, in contrast to the 83% observed inmosaic egg chambers containing mys PFCs (Fig. 4B,G). Second, weanalyzed whether halving the amount of Dl, by heterozygosity of a

Dl loss-of-function allele, which on its own did not cause any effect,would enhance the integrin phenotype. Indeed, we found that in 87%of mosaic egg chambers containing mys PFCs and heterozygous forDl (mys; Dl/+, n523), the mutant PFCs in contact with the germline

display differentiation defects, as monitored by FasIII expression(Fig. 4D, arrowhead). Third, we tested whether failure to maintaincontact with the germ line was, on its own, sufficient to affect proper

differentiation. To this end, we analyzed the expression of FasIII andEya in mosaic egg chambers containing bazooka (baz)-mutantfollicle cells, which very often grew extra layers at both poles of the

egg chamber. We did not find any alteration in the expression ofFasIII or Eya in the mutant cells located on the ectopic layers(Fig. 4E,F, arrows). And fourth, we tested whether increasing

integrin signaling without rescuing the multilayer phenotype wouldrescue the differentiation defects. To this end, we made use of achimeric integrin that allows constitutive integrin signaling inabsence of adhesion, the TorD/bcyt (Martin-Bermudo and Brown,

1999). We have previously shown that expression of this construct inmosaic egg chambers containing mys PFCs results in a partial rescue

Fig. 2. Defective endocycle and CycB expression in integrin-mutant PFCs located in ectopic layers. (A–D) S9–S10 wild-type (wt) and mosaic eggchambers were labeled with anti-GFP (green), anti-CycB (A,B; red), anti-Br-C (C,D; red) and TO-PRO-3 (blue). (A) In wild-type egg chambers, CycB isdownregulated at S6 (upper-left panel shows CycB expression in an S4 egg chamber). (B) However, this expression is maintained at S10 inmys PFCs located inectopic layers (GFP2, arrow). (C) In wild-type follicle cells, Br-C is upregulated after S6. (D) This upregulation fails to occur in mys PFCs located in ectopiclayers (GFP2, arrow). Note that in both cases mys PFCs in contact with the germline (GFP2, arrowhead in B and D) behave as wild-type follicle cells. (E,F) S11egg chambers stained with anti-GFP (green) and BrdU (red). Whereas in wild-type S11 egg chambers, BrdU incorporates in the four spots of amplification (E),genomic BrdU incorporation was observed in mys PFCs located in ectopic layers (F). (A9–F9) Magnifications of the white boxes in A–F, respectively.

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of the multilayer phenotype (Fernandez-Minan et al., 2007). Here,we found that it rescued the differentiation (Fig. 5C,D, n521) andnuclear size defects (Fig. 5E,G, n526) in 100% of the cases,

including those in which the stratification phenotype was notrescued. Taken together, these results strongly suggest that the celldifferentiation phenotype is not just a consequence of the

multilayering and that integrin-mediated signaling is required toenhance Notch signaling in PFCs.

Finally, we tested whether elimination of other focal adhesion

components, such as Talin, would also affect proper follicle celldifferentiation. In order to do this, we generated mosaic eggchambers containing follicle cells mutant for rhea, the geneencoding Talin in Drosophila (Brown et al., 2002). We have

previously shown that removal of rhea in follicle cells causespolarity and stratification defects identical to those caused by loss ofmys (Fernandez-Minan et al., 2008; Fig. 5F). However, we found

that both the expression of FasIII and the nuclear size of all rhea

PFCs, including those located in ectopic layers, were similar to thoseof wild-type PFCs (Fig. 5F,H). These results argue against the view

that the differentiation defects observed in mys PFCs are mainly dueto the multilayering and polarity phenotypes and strongly supportour conclusion that integrins strengthen Notch signaling.

The Notch intracellular domain can rescue the Notchsignaling phenotype of mys mutant PFCsTo investigate how integrin function regulates Notch signaling,

we tested whether the expression of various Notch constructs inmys PFCs could rescue the differentiation defects. As a result of

ligand binding, Notch is proteolytically processed, and theextracellular domain (NECD) is transendocytosed into theligand-sending cell. The remaining receptor undergoes two

successive proteolytic cleavages, first by ADAM and then by c-secretase, the second step requiring Notch endocytosis. This isfollowed by the release of the Notch intracellular domain (NICD)

and its translocation to the nucleus, where it regulates thetranscription of its downstream targets (reviewed in Fortini andBilder, 2009). We found that when the NICD was expressed in

mys PFCs, both the nuclear size defect and the differentiationphenotype were rescued, as FasIII expression after S6 was onlydetected in 13% of the egg chambers carrying mys PFCs inectopic layers (Fig. 6A, arrow), in contrast to the 83% observed

in mosaic egg chambers containing mys PFCs without NICD(Fig. 1E). Interestingly, neither full-length Notch nor NECD wereable to rescue the differentiation phenotype (Fig. 6B; data not

shown). This result suggests that elimination of integrins disruptsNotch signaling probably at the level of the final Notch cleavage,as this generates the functional NICD. This cleavage is regulated

at the level of endosomal trafficking. Thus, to further investigatethe consequences of eliminating integrin function on the Notchpathway, we compared the expression and localization pattern of

the Notch receptor itself in wild-type and mys PFCs. In wild-typefollicle cells up to S6, the Notch receptor accumulates at highlevels on the apical side. From this stage onwards, upon Notchactivation by Dl from the germ line, the levels of Notch in the

membrane are strongly reduced (Lopez-Schier and St Johnston,2001; Fig. 6C, arrowhead). In mys PFCs located in ectopic layers,

Fig. 3. The Notch pathway is disrupted in integrin-mutant PFCs. Egg chambers were stained with TO-PRO-3 (A–F; blue), anti-GFP (A–F; green), anti-Cut(A,B; red), anti-Hnt (C,D; red) and anti-b-galactosidase (E,F; red) to detect the reporter of Notch activity, Gbe-Su(H)-lacZ. In wild-type (wt) egg chambers, Notchsignaling induces downregulation of Cut (A) and upregulation of Hnt (C) from S6 onwards (upper-left panel in A shows the expression of Cut in an S4 eggchamber). By contrast, mys PFCs located in ectopic layers (GFP2, arrow) show prolonged Cut expression after S6 (B) and fail to upregulate Hnt at S7 (D). (E) Inwild-type egg chambers, expression of the reporter of Notch activity, Gbe-Su(H)-lacZ is upregulated after S6 in PFCs. (F) This upregulation fails to take place inmys PFCs in ectopic layers (GFP2, arrow). mys PFCs in contact with the germline (GFP2, arrowhead in D and F) behave as wild-type follicle cells.(A9–F9) Magnifications of the white boxes in A–F, respectively.

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we found significant accumulations of both NICD and NECD at orbelow the plasma membrane (Fig. 6D, n532; supplementary

material Fig. S3B,C, arrows), suggesting that full-length Notch ispresent in excess amounts in these mutant cells. Furthermore, partof the ectopic Notch protein was visible in a punctate cytoplasmic

pattern, indicating that some of the Notch protein was accumulatingin vesicles (white arrows in Fig. 6D9; supplementary material Fig.S3B,C). In fact, we found that several of the ectopic Notch-positivevesicles present in mys PFCs were also positive for the early

endosomal marker Hrs (hepatocyte growth factor-regulated tyrosinekinase substrate, Fig. 6E, arrow). This result suggested a potentialdefect in the processing and/or endocytic trafficking of Notch in

mys PFCs. The aberrant Notch distribution observed in mys PFCscould just be a consequence of the multilayering and/or polaritydefects found in mys PFCs. Alternatively, integrins might be

required for proper endosomal trafficking of Notch. To distinguishbetween these two possibilities, we performed two sets ofexperiments. First, we tested whether halving the amount of Dl,which, on its own, does not affect Notch distribution (data not

shown), affected Notch trafficking in mys PFCs that contacted thegermline. Indeed, we found a punctate distribution of Notch in the

cytoplasm of mys; Dl/+ PFCs in contact with the germline (Fig. 6F,arrowhead), which resembled that observed in mys PFCs located inectopic layers (Fig. 6D, arrow). And second, we analyzed Notch

distribution in baz follicle cells, which very often show bothmultilayering and polarity defects. We found that, in contrast towhat happens in mys PFCs, there is no increased accumulation ofNotch at or below the plasma membrane in baz PFCs located in

ectopic layers (supplementary material Fig. S3D). These resultsstrongly suggest that the changes in Notch distribution found in mys

PFCs are not just a consequence of the multilayering or polarity

defects and that integrins contribute to the proper endosomaltrafficking and/or processing of the Notch receptor.

Integrins are required for proper downregulation of theexpression of dacapo but not that of stringAn intriguing aspect of the ovarian phenotype of mys whencompared with that of Notch is that even though, like Notch,

Fig. 4. Integrins are required to strengthen Notch signaling. S9–S10 egg chambers were labeled with anti-GFP (green), anti-FasIII (A–E; red), anti-Eya(F; red) and TO-PRO-3 (blue). (A) In an H/+ control egg chamber, FasIII (red) is restricted to polar cells, as is the case in wild-type egg chambers. (B) S10 mosaicegg chambers showing the abnormal expression of FasIII (red) in mys PFCs located in ectopic layers. (C) Reducing the amount of H restores FasIII expressionand nuclear size in mys PFCs located in ectopic layers (GFP2, arrow). This is quantified in G and H, respectively. (D) mys PFCs in contact with the oocyte(arrowhead) show high levels of FasIII when Dl function is reduced. (E,F) FasIII (E) and Eya (F) expression patterns are not affected in baz PFCs located inectopic layers. (A9–F9) Magnifications of the white boxes in A–F, respectively. Data in G and H show the mean6s.d. wt, wild type.

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integrin mutant PFCs fail to enter endocycle and to differentiate,

in contrast to Notch mutants, they do not continue proliferating.The Notch pathway has been shown to regulate the mitotic-to-endocycle transition by independently regulating three mitoticregulators: it downregulates the G2 phosphatase String (Stg) and

the G1 CKI p21CIP/Dacapo (Dap) and upregulates the APCactivator Fzr (Shcherbata et al., 2004). To investigate what liesbehind the difference between the Notch and mys phenotypes, we

analyzed the expression of these cell cycle regulators in mys PFCsusing lacZ reporters (Lehman et al., 1999; Shcherbata et al.,2004). dap-lacZ and stg-lacZ are normally expressed in mitotic

follicle cells from S4 to S6, when they are downregulated(Lehman et al., 1999; Shcherbata et al., 2004; Fig. 7A,C).However, we found that even though mys PFCs in contact with

the germline showed no alteration of the dap-lacZ pattern ofexpression (arrowheads in Fig. 7B), those located in ectopiclayers showed prolonged expression of dap-lacZ (arrow inFig. 7B, 86%, n539) after S6. By contrast, all mys PFCs were

able to downregulate stg-lacZ (Fig. 7D, n530).Taken together, our results show that, when contact with the

germline is lost, integrins are required in PFCs to downregulate

the expression of the CKI dap but not that of string. This might

explain why mys PFCs do not differentiate properly, yet they donot continue dividing after S6. We next decided to test this byperforming two sets of experiments. First, we tested whether areduction in the amount of Dap would rescue the differentiation

and cell cycle defects found in mys PFCs located in ectopiclayers. Indeed, we found that removing one copy of dap partiallyrescued the differentiation and Notch signaling defects of mys

PFCs, as now only 33% (n526) and 35% (n524) of mosaic eggchambers containing mys PFCs and heterozygous for dap (mys;dap/+) showed expression of FasIII and Cut, respectively, after

S6 (arrows in Fig. 7E,F,I), in contrast to the 83% and 82% foundin mosaic egg chambers containing mys PFCs (Fig. 1E; Fig. 3B;Fig. 7I). In addition, the aberrant pattern of BrdU incorporation

and nuclear size of mys PFCs were also partially rescued(Fig. 7G,I,J), as now only 35% (n526) of mys; dap/+ eggchambers carrying mys PFCs showed an aberrant BrdUincorporation pattern, in contrast to the 75% observed in egg

chambers containing mys PFCs (Fig. 7G,I). Second, we analyzedwhether the expression of stg was sufficient to maintain mys PFCslocated in ectopic layers within the cell cycle after S6. Using the

Fig. 5. Integrin-mediated signalingis required for PFC maturation. Eggchambers were stained with TO-PRO-3 (E,F; blue), anti-GFP(A–F; green), anti-FasIII (A,C,F; red),anti-Cut (B,D; red) and anti-Myc(C,D; blue) to detect the TorD/bcytchimeric integrin. (A,B) S9–S10mosaic egg chambers showingabnormal expression of FasIII (A; red)and Cut (B; red) in mys PFCs locatedin ectopic layers (GFP2, arrow).(C–E) Ectopic expression of TorD/bcyt(blue) in these mys PFCs restores thenormal expression of FasIII (C; red),Cut (D; red) and nuclear size (E,G).(F) FasIII expression is not affected inrhea PFCs located in ectopic layers(GFP2, arrow). (G,H) Quantification ofthe nuclear size of the indicatedgenotypes. The nuclear size of rheaPFCs is similar to that of wild-type(wt) follicle cells (H). In all cases, mys

PFCs in contact with the germline(GFP2, arrowheads in A–E) behaveas wild-type follicle cells. Data showthe mean6s.d.

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anti-PH3 antibody, we found that indeed this was the case(Fig. 7H, arrow).

DISCUSSIONThe coordination of cell proliferation with the gradualdifferentiation of different cell types is essential for proper

development and tissue homeostasis. However, little is knownabout the signals that couple cell cycle exit to differentiationswitch during development. Here, we show that signalingmediated by integrins, the major cell–ECM receptors,

contributes to the regulation of this switch in the posteriorfollicle cells of the Drosophila ovary. Furthermore, ourexperiments strongly suggest that one of the mechanisms by

which integrins regulate epithelial cell differentiation is bymodulating the activity of the Notch pathway through promotingthe proper endosomal trafficking and/or processing of Notch.

Integrins are known to regulate cell differentiation andproliferation in other systems. The effects of particular integrinsin regulating differentiation vary depending on the epithelial celltype. Thus, although b1 integrin signaling is inhibitory for

differentiation in the epidermis (reviewed in Watt, 2002), it iscrucial for proper differentiation in the mammary gland (Nayloret al., 2005). Interestingly, as we show here for Drosophila PFCs,

loss of b1 integrin in the mammary gland leads to an upregulationin the expression of the CKI p21Cip1 (also known as CDKN1A)(Li et al., 2005). Furthermore, as is the case for PFCs, this

upregulation is responsible for the proliferation defects observedin b1-integrin-mutant mammary glands, because knockdown of

p21Cip1 rescues the effect of b1 integrin loss. Integrins have beenshown to control CKI levels in other cell types. Thus, b1-integrin-mutant chondrocytes show a defect in the G1–S transition, which

is accompanied by upregulation of the CKIs p16Ink4a (also knownas CDKN2A) and p21Cip1 (Aszodi et al., 2003). Ablation of b1integrin in developing cerebellum results in a failure in the

expansion of the cerebellar granule cell precursors due toupregulation of p27Kip1 (also known as CDKN1B) (Blaesset al., 2004). However, the mechanisms underlying thisregulation remain to be determined. In summary, our work

extends previous studies demonstrating a key role for integrinsignaling in controlling growth and differentiation by regulatingCKI levels, which suggests that this role is conserved throughout

evolution. In addition, our studies unravel one of the mechanismsunderlying this regulation – modulation of Notch signaling.

We have found that elimination of integrins results in defective

Notch signaling in follicle cells. However, one puzzling aspect ofthe integrin phenotype is the fact that the phenotypes of defectiveNotch are partial and position-dependent. Similar to Notch-mutant PFCs, integrin-mutant PFCs fail to enter endocycle and to

differentiate; however, unlike Notch-mutant cells, they do notcontinue proliferating. In addition, and in contrast to Notchmutants, the defects due to absence of integrin function are

restricted to PFCs. Position-dependent phenotypes have also beenobserved in follicle cells in mutants of the SWH pathway ormutants with excessive myosin activity (Meignin et al., 2007;

Polesello and Tapon, 2007; Sun et al., 2011; Yu et al., 2008).These position-dependent responses could be due to the

Fig. 6. Integrins modulate the Notch pathway by regulating its intracellular trafficking and/or processing. Egg chambers were stained with anti-GFP(A–F; green), anti-FasIII (A,B; red), anti-NICD (C–F; red), anti-Hrs (E; green) and TO-PRO-3 (blue). Expression of NICD (A) but not full-length Notch (‘N’; B)restores the normal expression of FasIII and nuclear size of mys PFCs in ectopic layers (GFP2, arrow). (C) NICD expression in wild-type (wt) eggchambers (arrowhead). (D) mys PFCs located in ectopic layers (GFP2, arrow) contain discrete cytoplasmic and membrane-associated accumulations of NICD.(E) Some of the ectopic NICD is found to colocalize with Hrs (arrow). Note that mys PFCs in contact with the germline show normal NICD localization(GFP2, arrowhead in D). (F) However, reduction of Dl function in these mutant PFCs (arrowhead) leads to an accumulation of NICD similar to that found in mys

PFCs in ectopic layers (arrow in D). (A9–F9) Magnifications of the white boxes in A–F, respectively.

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involvement of signaling pathways specific to these follicle cell

subpopulations, such as the EGFR and the JAK–STAT pathways.Alternatively, they could reflect an intrinsic difference in Notchsignaling levels between the PFCs and other follicle cells, as it

has been proposed that the expression of Dl in the oocyte is lowerthan that in the nurse cells (Yu et al., 2008). Therefore, theintensity of Notch signaling might be weaker in PFCs with

respect to other follicle cells and might depend more on positivemodulators such as integrins to achieve adequate levels of

activity. In this context, integrin signaling might play a general

role in reinforcing Notch activity, with this regulation being moreimportant on a sensitized background. This could explain therestriction of the Notch defects to integrin-mutant PFCs located in

the ectopic layers, as Notch signaling in these cells could berelatively low, given that they are at a greater distance from thesource of ligand. In addition, a role for integrins as positive

modulators of Notch signaling could also explain why dacapo butnot string expression is affected in integrin-mutant PFCs. The

Fig. 7. Integrins regulate PFC maturation by regulating dap expression. Egg chambers were stained with anti-GFP (A–H; green), anti-FasIII (E; red), anti-Cut (F; red), anti-BrdU (G; red), anti-PH3 (H; red), TO-PRO-3 (A–F,H; blue) and anti-b-galactosidase (A–D; red) to monitor the expression of the dap-lacZ

(A,B) and stg-lacZ (C,D) reporters. (A,C) In wild-type (wt) egg chambers, dap (A) and stg (C) are downregulated at S6. Upper-left panels show the expression ofthese markers in S4 egg chambers from the same ovarioles. (B,D) In mys PFCs located in ectopic layers (GFP2, arrow), dap expression is maintainedafter S6 (B) whereas stg is properly downregulated (D).mys PFCs in contact with the germline show normal dap and stg expression (GFP2, arrowheads in B andD). (E–G) Reducing dap levels in mosaic egg chambers carrying mys PFCs restores normal expression of FasIII (E,I) and Cut (F,I), BrdU incorporationpattern (G,I) and nuclear size (J). (H) Ectopic expression of stg in mys PFCs located in ectopic layers (GFP2, arrow) is sufficient to maintain these mutant cellswithin the cell cycle. (A9–H9) Magnifications of the white boxes in A–H, respectively. Data in I and J show the mean6s.d.

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reduced Notch activity present in the integrin-mutant PFCs mightbe sufficient to induce normal levels of string expression but not

of dacapo. In fact, Notch has been shown to independentlycontrol these two cell cycle regulators in follicle cells (Shcherbataet al., 2004).

There are different mechanisms by which integrins could

modulate Notch signaling. We have shown that defective Notchsignaling in mys PFCs can be rescued by expression of NICD,but not by full-length Notch or NECD. This suggests that

elimination of integrins disrupts Notch signaling, most likely atthe level of the last Notch cleavage, which generates thefunctional NICD. This step is subject to regulation at the level of

endosomal trafficking (reviewed in Fortini and Bilder, 2009). Infact, we have observed increased punctate distribution of Notchin the cytoplasm of integrin-mutant PFCs. Therefore, we

propose that a way by which integrins could modulate Notchactivity in PFCs could be through the regulation of itsintracellular trafficking and/or processing. At what level couldintegrins and Notch interact? A level of intersection between

Notch and integrins could be at the plasma membrane lipid rafts,specialized membrane microdomains involved in endocytosisand signaling (Lajoie and Nabi, 2010). Interestingly, mutations

in genes required for the biogenesis of sphingolipids, majorconstituents of lipid rafts, such as lace and acc, cause abnormalaccumulation of Notch in endosomes and significant alterations

in the pathway (Sasamura et al., 2013). These results have led tothe suggestion that genes involved in the formation and/orfunction of lipid rafts might influence tissue growth and

differentiation by regulating the intracellular trafficking ofsignaling molecules such as Notch. Integrin signaling has beenshown to regulate the location and behavior of lipid rafts (delPozo et al., 2004; Norambuena and Schwartz, 2011).

Furthermore, the local regulation of these membrane domainsby integrins has been proposed to locally regulate signalingproteins thought to associate with these domains, such as the

PDGFR or Rac (Baron et al., 2003). Thus, lipid rafts might be acellular platform to allow for Notch–integrin interactions.Finally, the increased levels of Hrs in cytoplasmic vesicles

observed in mys PFCs suggest that the endocytosis defects mightnot be specific to the Notch receptor, but rather might indicate amore generalized defect in endocytosis.

A temporal coupling of cell-cycle arrest and terminal

differentiation is common during development and homeostasis.Furthermore, a loss of coordination between these two processescan lead to aberrant tissue development and tumorigenesis. Here,

we have shown that, in Drosophila, as is the case in mammals,integrins can regulate the switch between cell cycle anddifferentiation in epithelial cells by regulating CKI levels,

suggesting that this role of integrins is conserved. Furthermore,we show that one of the mechanisms by which integrins regulatethe switch between cell cycle and differentiation in follicle cells

is by modulating the activity of the Notch pathway, and that thisregulation might be achieved by promoting proper endosomaltrafficking of Notch. As integrins transmit signals from the ECMinside the cell, our study demonstrates that the ECM environment

can fine-tune the cellular response to pathways regulating cellproliferation and differentiation, such as the Notch pathway, andultimately can determine the cellular state of a specific cell type.

In the future, it will be interesting to test whether integrins couldinteract with other signals known to regulate follicle celldifferentiation, such as those downstream of the SWH pathway

or myosin activity.

MATERIALS AND METHODSFly stocksTo generate follicle cell mutant clones we used the FRT/FLP technique

(Chou and Perrimon, 1992). Mutant clones were marked by the absence

of GFP. The following mutant alleles and chromosomes were

used: mys11 [also known as mysXG43 (Bunch et al., 1992)], e22c-Gal4

UAS-flipase (Duffy et al., 1998), H2 (Bang and Posakony, 1992),

dap04454-LacZ, DlB2 and Notch55e11 (Bloomington Stock Center), Gbe-

Su(H)-lacZ (Furriols and Bray, 2001), pnt-LacZ (Morimoto et al., 1996),

UAS-TorD/bcyt (Martin-Bermudo and Brown, 1999), baz815 (Djiane

et al., 2005), rhea79 (Brown et al., 2002) and ilk1 (Zervas et al., 2001).

The e22c-Gal4 driver is expressed in the follicle stem cells in the

germarium and was therefore used in combination with UAS-flp to

generate large mys and Notch follicle cell clones. The GR1Gal4 is

expressed in all follicle cells (Gupta and Schupbach, 2003) and it was

used to express N full-length, NEXT, NICD (Rauskolb et al., 1999) or

string (Bloomington Stock Center) in mys PFCs in combination with the

HS flipase system. Thus, mys11FRT101/FMZ; UAS-N full length (or

UAS-NICD or UAS-NEXT) were crossed to y w hs-Flipase 122

ubiquitin-GFPFRT101;; GR1Gal4 males. Newly hatched females from

this cross were heat-shocked at 37 C for 20 minutes and kept at 25 C for

2 days prior to ovary dissection. To generate mys mutant cells

expressing TorD/bcyt (Martin-Bermudo and Brown, 1999) in the

follicular epithelium, we grew females of the genotype mys11FRT101/

ubiquitin-GFPFRT101;e22c-Gal4/UAS-TorD/bcyt; tubulin-Gal80ts/+ at

18 C until eclosion. Adult females were kept at 31 C for 3–5 days prior

to ovary dissection. To generate clones of cells expressing CycE, Stg,

DP and E2F2 in the follicular epithelium, we used the ‘flip-out’

technique (Struhl et al., 1993). y w hs-Flipase 122, Act ,CD2

.Gal4;;UAS-GFP females were crossed to UAS-CycE,UAS-Stg;UAS-

DP,UAS-E2F2 (Buttitta et al., 2007). Newly hatched females from this

cross were heat-shocked at 37 C for 20 minutes and kept at 25 C for

2 days prior to ovary dissection.

Immunohistochemistry and microscopyDrosophila females were yeasted for 1–2 days before dissection. Staining

was performed at room temperature according to standard procedures. The

DNA dye TO-PRO-3 (Molecular ProbesTM, 1:1000) was added for

10 minutes in PBT (PBS+0.1% Tween) after the secondary antibody.

The following primary antibodies were used: mouse anti-FasIII (1:20),

mouse anti-Eya (1:20), mouse anti-Cut (1:25), mouse anti-NICD (1:100),

mouse anti-NECD (1:100), mouse anti-Hnt (1:15), mouse anti-CycB (1:20)

and mouse anti-Br-C (1:100; all from Developmental Studies Hybridoma

Bank; DSHB); rabbit anti-Staufen (1:1000) (St Johnston et al., 1991);

mouse anti-BrdU (1:20; Roche); mouse anti-b-gal (1:10,000; Promega);

rabbit anti-b-gal (1:10,000; CAPPELTM); rabbit anti-GFP (1:10,000;

Molecular ProbesTM); mouse anti-GFP (1:100; Molecular ProbesTM);

mouse anti-Myc (1:100; Oncogen Science) and guinea-pig anti-Hrs

(1:1000; Lloyd et al., 2002). The secondary antibodies were conjugated

to Alexa Fluor 488 (Molecular ProbesTM), Cy3 or Cy5 (Jackson

ImmunoReseach Laboratories) and were used at 1:200. Images were

captured with a Leica TCS-SPE confocal microscope and processed with

ImageJ.

For the BrdU incorporation assay, the experiment was performed

using the 5-bromo-29-deoxy-uridine (BrdU) Labeling and Detection

Kit I from Roche (Cat. 11296736001). Ovaries were dissected in PBS

and incubated in BrdU incorporation medium (PBS+10 mM BrdU,

Roche) for 30 minutes and washed for 5 minutes with PBS. After

fixing the ovaries for 20 minutes in 4% paraformaldehyde (PFA), they

were blocked with PBT-10 (PBS+0.1% Tween, 10% BSA) for 1 hour.

Ovaries were then incubated overnight with anti-GFP antibody in

PBT-1 (PBS+0.1% Tween, 1% BSA) to label the clones, and were

washed with PBT-1 for 1 hour. Ovaries were re-fixed in 4% PFA for

20 minutes, treated with 4 M HCl in PBS for 7 minutes and washed

several times for 30 minutes in PBS, until the pH reached 7. Then,

ovaries were blocked again with PBT-10 and incubated overnight with

mouse anti-BrdU (Roche) diluted in incubation buffer. After washes

with PBT-1 for 1–2 hours, egg chambers were incubated with the

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secondary antibody in PBT-0.1 (0.1% Tween+0.1% BSA) for 2–

4 hours in the dark. Egg chambers were then washed with PBT for 1–

2 hours and finally mounted in Vectashield (Vector).

AcknowledgementsWe thank Sarah Bray, David Bilder, Sol Sotillos and Bloomington Stock Center forfly stocks and for sharing valuable reagents. Special thanks to Acaimo Gonzalez-Reyes and Sarah Bray for helpful comments on the manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsM.D.M.-B. conceived of the project, performed experiments, analyzed the dataand wrote the paper. M.J.G.-L., L.C.-R. and B.I.-J. performed experiments,analyzed the data and contributed to the writing of the paper. I.M.P. contributed tothe writing of the paper.

FundingThis work was supported by the Spanish Ministerio de Ciencia y Tecnologıa [grantnumbers BMC2010-16669, CSD-2007-00008 to M.D.M.-B.]; the Junta deAndalucıa (JA) [grant numbers CVI-280 and P09-CVI-5058 to M.D.M.-B.]; and theUK Medical Research Council (MRC) [grant number 200937 to I.M.P.]. M.J.G-L issupported by a FPI studentship from the Ministerio de Ciencia y TecnologUa.Institutional support from JA to C.A.B.D. is acknowledged. Deposited in PMC forrelease after 6 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.153122/-/DC1

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