ARTICLE

Fgf10 Maintains Notch Activation, StimulatesProliferation, and Blocks Differentiation ofPancreatic Epithelial CellsAlan Hart, Stella Papadopoulou, and Helena Edlund*

The pancreas is an endodermally derived organ that initially appears as a dorsal and ventral protrusion of the primitivegut epithelium. The pancreatic progenitor cells present in these early pancreatic anlagen proliferate and eventuallygive rise to all pancreatic cell types. The fibroblast growth factor receptor (FGFR) 2b high-affinity ligand FGF10 hasbeen linked to pancreatic epithelial cell proliferation, and we have shown previously that Notch signalling controlspancreatic cell differentiation by means of lateral inhibition. In the developing pancreas, activated intracellular Notchappears to be required for maintaining cells in the progenitor state, in part by blocking the expression of thepro-endocrine gene neurogenin 3 (ngn3), and hence endocrine cell differentiation. Here, we show that persistentexpression of Fgf10 in the embryonic pancreas of transgenic mice also inhibits pancreatic cell differentiation, whilestimulating pancreatic epithelial cell proliferation. We provide evidence that one of the effects of the persistentexpression of Fgf10 in the developing pancreas is maintained Notch activation, which results in impaired expressionof ngn3 within the pancreatic epithelium. Together, our data suggest a role for FGF10/FGFR2b signalling in regulationof pancreatic cell proliferation and differentiation and that FGF10/FGFR2b signalling affects the Notch-mediatedlateral inhibition pathway. Developmental Dynamics 228:185–193, 2003. © 2003 Wiley-Liss, Inc.

Key words: pancreas; fgf10; epithelial cells; proliferation, notch

Received 12 May 2003; Accepted 19 June 2003

INTRODUCTION

Pancreatic islet cell transplantationis today an established procedure torestore the required mass of func-tional �-cells and long-term normo-glycemia in Type 1 diabetic patients(Ryan et al., 2001; Shapiro et al.,2000), and this therapy could be anattractive option also for patientswith severe type 2 diabetes. The useof this therapy is, however, restricteddue to a shortage of transplantable

material. One attractive alternativesource for transplantable �-cells as acure for diabetes involves the gen-eration of functional �-cells fromstem and/or progenitor cells. Apartfrom the identification and isolationof suitable stem or progenitor cells,being of embryonic, fetal, or adultorigin, the factors that allow us toexpand and ultimately induce thedifferentiation of these stem/pro-genitor cells into functional �-cells,

must be identified. Hence, to realisethe full potential of stem cells, themolecular mechanisms controllingthese processes need to be eluci-dated. Throughout development,cell proliferation and differentiationhave to be carefully regulated toensure proper generation of func-tional organs and tissues. Such de-velopmental processes are con-trolled, temporally and spatially, bymeans of various signalling path-

Umeå Center for Molecular Medicine (UCMM), University of Umeå, Umeå, SwedenThe Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/developmentaldynamics/suppmat/index.htmlGrant sponsors: Swedish Research Council; Juvenile Diabetes Foundation, New York; Wallenberg Consortium North; EU 5th program; EUregional fund, objective 1.Drs. Hart and Papadopoulou contributed equally to this work.*Correspondence to: Helena Edlund, Umeå Center for Molecular Medicine (UCMM), University of Umeå, SE-901 87 Umeå, Sweden.E-mail: helena.edlund@ucmm.umu.se

DOI 10.1002/dvdy.10368Published online 25 August 2003 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 228:185–193, 2003

© 2003 Wiley-Liss, Inc.

Fig. 1. Fibroblast growth factor receptor(FGFR) 2 is expressed in the developingpancreatic epithelium. A–F: Confocal anal-yses of the expression of FGFR2 at embry-onic day (e) 9.5 (A), e11.5 (B), e13 (C), e15(D), neonatal (E, neo), and adult (F) stagesshow that FGFR2 is expressed in pancreaticprogenitor cells at early stages of develop-ment but then becomes gradually re-stricted to pancreatic �-cells. Glu, gluca-gon; Ins, insulin. C applies to Scale bars �0.04 mm in C applies to A–D, 0.03 mm in F.

Fig. 2. Pancreatic hyperplasia in Ipf1/Fgf10 mice. A–D: Gross morphologic anal-yses of embryonic day (e) 15 (A,B) andneonatal (C,D) wild-type (A,C), and Ipf1/Fgf10 transgenic (B,D) mice demonstratingthe enlarged pancreas, more tightlypacked appearance of the transgenicpancreas compared with that of stage-matched wild-type littermates. The size ofthe pancreata in A and B is indicated bythe dotted lines. St, stomach; Sp, spleen;Dp, dorsal pancreas; Vp, ventral pancreas;d, duodenum.

Fig. 3. The pancreas of Ipf1/Fgf10 micedisplays a condensed epithelial structure.A–L: Histologic analyses of embryonic day(e) 13 (A,D,G,J), e17 (B,E,H,K), and neonatal(postnatal day 1 [P1], C,F,I,L), wild-type (A–C,G–I), and Ipf1/Fgf10 transgenic (D–F,J–L)mice, by using DAPI (A–F) and hematoxy-lin–eosin (G–L) staining demonstrating thecompact, dense epithelial structure of thetransgenics compared with that of wild-types. Scale bars � 0.04 mm in A (applies toA–L).

ways. The pancreas develops bymeans of evaginations of the primi-tive gut epithelium resulting in theformation of the dorsal and ventralpancreatic anlagen. The pancre-atic progenitor cells present in theseearly pancreatic anlagen coexpressthe transcription factors IPF1/PDX1,Nkx2.2, Nkx6.1, and p48 (Chiangand Melton, 2003), and subse-quently proliferate and differentiatethus eventually giving rise to all dif-ferentiated, pancreatic epithelialcell types present in the mature pan-creas (Edlund, 2002).

We have identified previously theNotch-signalling pathway as amechanism through which pancre-atic endocrine cell fate is regulated(Apelqvist et al., 1999). Studies onHes-1 (Jensen et al., 2000), neuroge-nin (ngn) 3 (Gradwohl et al., 2000),Dll-1, and RBP-J�-deficient mice(Apelqvist et al., 1999), as well asmice overexpressing ngn3 in earlypancreatic progenitors (Apelqvist etal., 1999; Schwitzgebel et al., 2000),collectively demonstrate that Notchsignalling controls the choice be-tween differentiated endocrine andprogenitor cell fates in the develop-ing pancreas. These data also re-veal that ngn3 not only is competentto promote, but also required for,pancreatic endocrine cell differenti-ation. During pancreatic cell devel-opment, impaired Notch receptoractivation or signalling results in pro-found ngn3 gene expression, leadingto premature endocrine cell differen-tiation at the expense of pancreaticcell expansion and exocrine cell dif-ferentiation (Apelqvist et al., 1999;Jensen et al., 2000). In contrast, cellswith active Notch-signalling mostlikely remain as undifferentiated pro-genitor cells that would allow the sub-sequent proliferation, morphogenesis,and later differentiation of pancreaticepithelial cells analogous to the func-tion of Notch-signalling during earlymammalian neurogenesis (Lewis,1996; Beatus and Lendahl, 1998).

FGF-signalling plays a key role inthe development of the mouse em-bryo and has been implicated in thedevelopment of many organs thatare dependent on epithelial–mes-enchymal interactions (Kato andSekine, 1999; Szebenyi and Fallon,1999). The pancreas is an organ

whose growth, branching morpho-genesis, and differentiation are de-pendent on epithelial–mesenchy-mal interactions, thus implicating apotential role for FGFs and/or othergrowth factors (Edlund, 2002). Wehave shown previously that FGFR1csignalling in the adult mouse �-cells isrequired for normal �-cell functionand maintenance of normoglyce-mia (Hart et al., 2000), and otherstudies have suggested a role forFGF-signalling during pancreatic de-velopment (Celli et al., 1998; Miralleset al., 1999; Ohuchi et al., 2000; Bhus-han et al., 2001; Revest et al., 2001;Elghazi et al., 2002). Mice that ex-press a dominant negative FGFR2bunder the control of the Metallothio-nein promoter (Celli et al., 1998),and mice that lack FGFR2b (Revestet al., 2001) or Fgf10 (Ohuchi et al.,2000; Bhushan et al., 2001), a high-affinity ligand for FGFR2b, all showpancreatic hypoplasia. In vitro cul-tures of rat pancreatic anlagenhave suggested that signalling bymeans of the FGFR2b stimulates exo-crine differentiation and pancreaticendocrine progenitor cell prolifera-tion (Miralles et al., 1999; Elghazi etal., 2002). We here show that persis-tent expression of the FGFR2b high-affinity ligand Fgf10 in the develop-ing pancreatic epithelium oftransgenic mice results in enhanced,prolonged proliferation of pancre-atic epithelial cells, pancreatic hy-perplasia, and impaired pancreaticcell differentiation. Moreover, ourfindings suggest that the effects ex-erted by Fgf10 perturb the lateral inhi-bition process and maintains Notchactivation throughout the pancreaticepithelium.

RESULTS

Perturbed PancreaticDevelopment in Ipf1/Fgf10Transgenic Mice

FGFR2 was expressed in nondifferen-tiated epithelial pancreatic cells be-tween embryonic day (e) 9 and e13but not in differentiated endocrinecells that appear at these stages(Fig. 1A–C). At later embryonicstages, the expression of FGFR2 be-comes restricted such that by theneonatal stage, continuing through

to the adult, the expression is limitedto insulin-producing cells (Fig. 1D–F).The FGFR2b high-affinity ligand Fgf10is highly expressed in the mesen-chyme surrounding the pancreaticbuds at early stages of development(Bhushan et al., 2001). We also ob-served a low-level Fgf10 expressionwithin the pancreatic epithelium atlater embryonic stages (Supplemen-tary Fig. 1, which is available online athttp://www.interscience.wiley.com/developmentaldynamics/suppmat/index.html). To begin to define themechanism by which FGF10/FGFR2signalling controls pancreatic progen-itor cell proliferation and differentia-tion, we here used the Ipf1/Pdx1 pro-moter to generate transgenic miceexpressing Fgf10 within the pancre-atic epithelium throughout pancre-atic development.

Transgene expression was con-firmed by in situ hybridisation and re-al-time polymerase chain reaction(PCR; Supplementary Fig. 1 anddata not shown) and these expres-sion analyses also showed that thepancreatic expression of Fgf10 wasincreased two- to fourfold in trans-genic mice compared with that ofnormal mice. Ipf1/Fgf10 transgenicmice were born alive and consis-tently smaller in size compared withtheir control littermates (data notshown). The transgenic pups ap-peared dehydrated, and the major-ity died at postnatal day (P) 1 orwithin the first postnatal week. Mea-surement of blood glucose levels ofP1–P3 neonates revealed elevatedblood glucose levels indicative ofhyperglycemia in the transgenic pups(data not shown). Dissections of 15-day-old embryos and neonatesshowed a macroscopically mal-formed pancreas that appearedgreatly increased in size comparedwith wild-type littermates (Fig. 2A–D).Apart from the overall, enlarged sizeof the pancreas, it also appearedmore solid and condensed, unlike thecharacteristic loose and fluffy struc-ture observed in pancreas dissectedfrom wild-type littermates (Fig. 2A–D).These findings show that persistent,high-level expression of Fgf10 in thedeveloping pancreas results in pan-creatic hyperplasia.

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Fig. 4. Enhanced proliferation of pancre-atic epithelial cells in Ipf1/Fgf10 mice. A–F:Cytokeratin-7 (A,C,D,F) and phospho His-tone H3 (PH3, B,C,E,F) immunostaining of em-bryonic day (e) 17 wild-type (A–C) and Ipf1/Fgf10 transgenic (D–F) pancreata reveal aperturbed organization of the transgenic ep-ithelium and an increased pancreatic epi-thelial cell proliferation in Ipf1/Fgf10 com-pared with wild-type mice. Scale bar � 0.05mm in A (applies to A–F).

Fig. 5. Impaired pancreatic cell differentia-tion in Ipf1/Fgf10 mice. A–L: Immunohisto-chemical analyses of differentiated pancre-atic markers in wild-type (A,C,E,G, I,K,) andIpf1/Fgf10 transgenic (B,D,F,H,J,L) neonatesshow in contrast to control mice only occa-sional cells expressing pancreatic endo-crine markers such as glucagon (Glu, A,B),insulin (Ins, A,B,I,J), and ISL1 (C,D) were de-tected in the transgenic pancreas. Car-boxypeptidase A (CPA, E,F) expression wasperturbed in the transgenic pancreas, whichalso lacked distinct acinar structures. A lowbut uniform expression of IPF1 (G,H), andNkx6.1 (I,J) appeared throughout the trans-genic pancreas compared with the strongand �-cell–specific expression seen in thewild-type pancreas. The transgenic pancre-atic epithelium also expressed Ptf1a/p48(p48, K,L). Scale bars � 0.04 mm in A (appliesto A–D,G–L), 0.05 mm in E (applies to E,F).

Fig. 6.

188 HART ET AL.

Progressive PancreaticHyperplasia in Ipf1/Fgf10Transgenic Mice

The increase in pancreatic cellulardensity in the transgenic micecompared with that of stage-matched wild-type was furtherdemonstrated by histologic analy-ses involving the visualization of nu-clei by DAPI, which confirmed theincreased cellular density in theIpf1/Fgf10 transgenic comparedwith stage-matched wild-type lit-termates (Fig. 3A–F). At e13, nodrastic difference in the structureor cellular density of the transgeniccompared with the wild-type pan-creas was discernible (Fig. 3A,D).By e17, the Ipf1/Fgf10 transgenicpancreas appeared, however, notonly increased in size comparedwith stage-matched littermates butalso displayed a more dense pan-creatic epithelial structure (Fig.3B,E) and the difference both insize and epithelial structure was fur-ther pronounced at the neonatalstage (Fig. 3C,F). Hematoxylin-eo-sin (HE) staining of different stagesof Ipf1/Fgf10 transgenic and wild-type pancreata confirmed the in-creasingly condensed structure ofthe pancreatic epithelium in thetransgenic mice and also suggestthat the transgenic epithelium isimmature with poorly developedacinar structures (Fig. 3G–L andSupplementary Fig. 2, available onlineat http://www.interscience.wiley.com/developmentaldynamics/suppmat/index.html). The increase in overall

size and the condensed structure ofthe transgenic pancreatic epitheliaat stages later than e13 show thatpersistent, high-level FgfF10 expres-sion results in progressive pancreatichyperplasia.

Increased Proliferation ofPancreatic Epithelial Cells inIpf1/Fgf10 Mice

To determine the extent of epithelialcell proliferation in the Ipf1/Fgf10mice compared with that of con-trols, we next performed double-immmunohistochemical analyses byusing antibodies against the ductal-specific marker cytokeratin-7 andantibodies specific for the mitoticmarker phospho-Histone H3 on e17pancreata (Fig. 4A–F). In the Ipf1/Fgf10 mice, cytokeratin-7 was ex-pressed throughout the epithelium incontrast to the more restricted,patchy expression observed in thewild-type pancreas (Fig. 4A,D andSupplementary Fig. 3, available on-line at http://www.interscience.wiley.com/developmentaldynamics/suppmat/index.html). Mitotically ac-tive phospho-H3–positive cells werefound uniformly throughout the e17Ipf1/Fgf10 pancreatic epithelium,whereas only a few scattered phos-pho-H3–positive cells were detectedin the pancreata of control mice.The number of phospho-Histone H3–positive cells/pancreatic area wasincreased by 50% e17 Ipf1/Fgf10mice compared with stage-matched controls, and the increasein the total pancreatic area in thetransgenic mice at this stage was�30% compared with controls. To-gether these data provide evidencethat the pancreatic hyperplasia ob-served in the Ipf1/Fgf10 transgenicmice results from an enhanced pro-liferation of the pancreatic epithelialcells.

Impaired Differentiation ofPancreatic Cell Types in Ipf1/Fgf10 Mice

To analyze in detail the differenti-ated state of the transgenic pan-creas at the neonatal stage, wenext examined the expression oftranscription factors, hormones,

and enzymes. Immunohistochemi-cal analyses of neonatal pancreasby using antibodies specific for in-sulin (Ins), glucagon (Glu), and so-matostatin (data not shown) con-firmed that pancreatic endocrinecell differentiation was severelyperturbed in the transgenic mice(Fig. 5A,B). In normal mice, endo-crine cells cluster into distinct islets(Fig. 5A). The Ipf1/Fgf10 transgenicmice displayed drastically fewer(less than 1% of that of wild-typepancreas) hormone-producingcells that failed to cluster (Fig. 5B).Consequently, the expression ofthe transcription factor ISL1, amarker for differentiated pancre-atic endocrine cells (Edlund, 2002),was mainly expressed in few, non-clustered cells corresponding tothe few hormone-producing cellsthat formed in the transgenic pan-creata, as opposed to the expres-sion detected in the clustered pan-creatic endocrine cells of controlpancreata (Fig. 5C,D). Togetherthese data provide evidence thatpancreatic endocrine cell differen-tiation is perturbed in Ipf1/Fgf10transgenic mice. The expression ofexocrine enzymes such as car-boxypeptidase A (Fig. 5E,F) was re-duced in Ipf1/Fgf10 mice com-pared with that observed in controlpancreata. The condensed, ab-normal organization of the pancre-atic epithelia, perturbed pancre-atic acinar formation, uniformcytokeratin-7 expression, and di-minished exocrine marker expres-sion in Ipf1/Fgf10 mice comparedwith control mice (Figs. 3, 5E,F) sug-gest that differentiation of pancre-atic epithelial cells into exocrinecells also is perturbed in Ipf1/Fgf10mice. Taken together, the in-creased pancreatic epithelial cellproliferation and impaired pancre-atic cell differentiation observed inIpf1/Fgf10 mice raise the possibilitythat the majority of these pancre-atic epithelial cells may representpancreatic progenitors.

Ipf1/Fgf10 Pancreatic EpithelialCells Express PancreaticProgenitor Cell Markers

Single-cell transcriptional profiling ofearly pancreatic epithelial cells

Fig. 6. The expression of the pro-endocrinemarker ngn3 expression is drastically per-turbed in Ipf1/Fgf10 transgenic mice. A–J: Insitu hybridisation analyses of embryonicday (e) 15 wild-type (A,C,E,G,I) and Ipf1/Fgf10 transgenic (B,D,F,H,J) pancreatashow that ngn3 (A,B) expression is severelyimpaired in the transgenic mice. Dll1 (C,D)expression in Ipf1/Fgf10 transgenic mice isalso reduced compared with that of stage-matched wild-type pancreas, whereasNotch1 (E,F) and HES-1 (G,H) is expressedthroughout the ductal epithelium of thetransgenic pancreas. Sel-1l (I,J) is stronglyexpressed in the forming acinar structuresof e15 wild-type pancreas, whereas Sel-1lexpression is reduced in the pancreatic ep-ithelium of Ipf1/Fgf10 transgenic mice.Scale bar � 0.05 mm in A–J.

Fgf10 AND PANCREATIC EPITHELIAL CELLS 189

(Chiang and Melton, 2003) togetherwith the expression profile of differ-ent pancreatic transcription factors(Edlund, 2002) suggests that earlypancreatic stem and/or progenitorcells coexpress the transcription fac-tors IPF1/PDX1, Nkx6.1, Nkx2.2, andp48. The transcription factor IPF1/PDX-1 is normally highly expressed inearly pancreatic progenitor cells, atlower levels in epithelial cells duringthe phase of massive pancreaticepithelial cell expansion (i.e., from�e10 and onward), and then be-comes highly expressed in mature�-cells with barely detectable ex-pression in other differentiated pan-creatic cell types (Ohlsson et al.,1993; Edlund, 1998). Ipf1/Fgf10 neo-nates displayed a uniform pancre-atic epithelial expression of IPF1/PDX1, reminiscent of the lower levelexpression of IPF1/PDX1 in proliferat-ing epithelial cells (Ohlsson et al.,1993; Edlund, 1998) with only occa-sional high-level IPF1/PDX1-express-ing cells (Fig. 5H). In control mice,IPF1/PDX1 was highly expressed inthe �-cells with only a low, barelydetectable expression in other pan-creatic cell types (Fig. 5G). The Nkx-transcription factors Nkx6.1 (Fig. 5I,J)and Nkx2.2 (not shown), which areexpressed in the early pancreaticprogenitor cells and later becomerestricted to �-cells (Nkx6.1), or allendocrine cells with the exceptionof �-cells (Nkx2.2; Sussel et al., 1998;Sander et al., 2000), were also ex-pressed at a low level in Ipf1/Fgf10pancreatic epithelial cells. Occa-sional cells expressing high levels ofthese transcription factors, repre-senting the few differentiated endo-crine cells that still appear in the Ipf1/Fgf10 mice, could also be observed inthe transgenic pancreas (data notshown). The pancreatic transcriptionfactor Ptf1a/p48 (Krapp et al., 1998),which is expressed in early pancreaticprogenitor cells from �e10 (Selanderand Edlund, 2002; Kawaguchi et al.,2002) and later becomes restricted todifferentiated exocrine cells (Krapp etal., 1998), was also expressed through-out the pancreatic epithelium oftransgenic neonates (Fig. 5K,L). Theexpression of IPF1/PDX1, Nkx6.1,Nkx2.2, and P48 in Ipf1/Fgf10 pancre-atic epithelial cells is supportive of the

immature, progenitor-like nature ofthese cells.

Expression of Notch-SignallingComponents Is Perturbed inIpf1/Fgf10 Mice

To investigate whether the impairedcell differentiation in the Ipf1/Fgf10mice reflected a perturbation in theinduction of pancreatic cell types ormerely a block in their terminal differ-entiation, we next analyzed the ex-pression of Notch signalling compo-nents in the transgenic pancreata.During pancreatic development,ngn3 expression marks endocrine pro-genitors and its expression is regulatedby means of the Notch-signallingpathway (Apelqvist et al., 1999;Jensen et al., 2000). Analyses re-vealed that ngn3 expression was im-paired in Ipf1/Fgf10 mice not only atneonatal stages (data not shown) butalso at e15 (Fig. 6A,B) compared withstage-matched littermates, suggest-ing that pancreatic endocrine cell dif-ferentiation is impaired already at theendocrine progenitor stage. Consis-tent with the reduced number ofngn3 expressing cells, Dll1 expressionalso appeared reduced comparedwith wild-type pancreas (Fig. 6C,D).No increased cell apoptosis was ob-served as determined by TdT-medi-ated dUTP nick-end labeling (TUNEL)assay or by staining for the apoptoticmarker Caspase 3 (data not shown),suggesting that the decreased num-ber of pro-endocrine and endocrinecells in Ipf1/Fgf10 mice is not causedby cell death but rather by impairedspecification of pro-endocrine cells. Inthe wild-type pancreas, Notch1 washighly expressed predominantly in thedeveloping acini at e15 (Fig. 6E,F) butabsent from the streaks of differentiat-ing endocrine cells (data not shownand Apelqvist et al., 1999). In the Ipf1/Fgf10 mice, Notch1 expression ap-peared to be expressed throughoutthe pancreatic epithelium, reminis-cent of the expression of Notch1 ine13 pancreatic epithelium (Apelqvistet al., 1999). The uniform expression ofNotch1 in the Ipf1/Fgf10 pancreaticepithelia was matched by the expres-sion of HES-1 (Fig. 6G,H), suggestingthat Notch activation is maintainedthroughout the pancreatic epitheliumof Ipf1/Fgf10 mice. A uniform, main-

tained activation of Notch activationis consistent with the impaired pan-creatic expression of ngn3 in Ipf1/Fgf10 mice. Together these data showthat persistent expression of Fgf10 im-pairs the expression of the pancreaticpro-endocrine gene ngn3, which inturn suggests that the Notch-medi-ated lateral inhibition pathway is im-paired in the Ipf1/Fgf10 mice. The lackof differentiated cell types in the Ipf1/Fgf10 mice might reflect a deregula-tion of Notch-mediated lateral inhibi-tion pathway where sustained Notchactivation would “lock” a majority ofthe pancreatic epithelial cells in anondifferentiated, progenitor-likestate with maintained proliferative ca-pacity.

Genes that regulate Notch activ-ity include sel-1, which in C. eleganshas been shown to act as a nega-tive regulator of Notch activity(Grant and Greenwald, 1997). Sel-1l,the mouse homologue (Donoviel etal., 1998) of C. elegans sel-1, starts tobe expressed in the developingpancreatic acini from �e13, andSel-1l remains highly expressed inexocrine cells at later stages of de-velopment and in the adult pan-creas (Fig. 6I and data not shown;Donoviel et al., 1998), implicating arole for Sel-1l during pancreatic celldifferentiation. In contrast to thehigh level of pancreatic expressionof Sel-1l in the developing acini ofe15 control mice, Sel-1l was ex-pressed at an apparently lower levelin the pancreas of e15 Ipf1/Fgf10transgenic mice (Fig. 6J). Togetherthese findings suggest that persistentductal epithelial expression of Fgf10perturbs the expression of the Notchantagonist Sel-1l.

DISCUSSION

Here, we show that persistent ex-pression of the FGFR2b high-affinityligand Fgf10 in the developing pan-creatic epithelium of transgenicmice leads to pancreatic hyperpla-sia and that the pancreatic epithe-lium of these mice is largely imma-ture. The gain-of-function datapresented here and the previouslyreported pancreatic hypoplasia ofmice lacking Fgf10 (Ohuchi et al.,2000; Bhushan et al., 2001) or per-turbed FGFR2b function (Celli et al.,

190 HART ET AL.

1998; Revest et al., 2001) togetherstrongly support a key role for FGF10/FGFR2 signalling in stimulating pan-creatic epithelial cell expansion.

Our data provide evidence thatFgf10 stimulates enhanced, pro-longed proliferation of pancreaticepithelial cells, and impairs pancre-atic cell differentiation. Moreover,our findings suggest that one of theeffects exerted by Fgf10 involves aperturbation of the lateral inhibitionprocess such that Notch activation ismaintained throughout the pancre-atic epithelium. The impaired ex-pression of the pro-endocrine genengn3 is consistent with a maintainedNotch activation and provides anexplanation for the perturbed differ-entiation of pancreatic endocrinecells observed in the transgenicmice. The condensed, atypical or-ganization of the pancreatic epithe-lia in Ipf1/Fgf10 mice together withthe uniform expression of cytokera-tin-7 and impaired expression of exo-crine enzymes show that pancreaticexocrine differentiation also is per-turbed in these mice. In addition tocytokeratin-7, the majority of thecells in the pancreatic epithelium ofthe Ipf1/Fgf10 mice express Ptf1a/p48, low levels of IPF1/PDX1, Nkx6.1and Nkx2.2, i.e., transcription factorsthat are expressed in pancreaticprogenitor cells (Chiang and Mel-ton, 2003). These cells do not expressendocrine markers like Isl1 and, al-though IPF1/PDX1 and Nkx6.1 areexpressed also in differentiated�-cells, Ptf1a/p48 is not. Taken to-gether, the impaired pancreatic celldifferentiation, pancreatic ductalhyperplasia, sustained pancreaticcell proliferation, and the expressionof pancreatic progenitor cell mark-ers in the transgenic pancreatic ep-ithelium, suggest that the proliferat-ing pancreatic epithelial cells of theIpf1/Fgf10 mice may possess pro-genitor-like properties.

The observation that Notch acti-vation appears to be maintained inthe pancreatic epithelium of Ipf1/Fgf10 mice suggests that the effectof FGF10/FGFR2b signalling on pan-creatic epithelial cell proliferationand differentiation involves theNotch signalling pathway. Cells withactivated Notch would be inhibitedto differentiate and instead main-

tain the ability to proliferate, result-ing in a sustained pancreatic epithe-lial cell proliferation throughoutpancreatic development. The rolefor Fgf10 during pancreatic devel-opment might thus be dual; to main-tain the pancreatic progenitor cellsin an undifferentiated state and toprovide the proliferative cue. Duringnormal pancreatic developmentFgf10 is predominantly expressed inthe mesenchyme and as pancre-atic development progresses themesenchyme/epithelium ratio de-creases. Hence, at later stages ofpancreatic development the con-centration of Fgf10 ligand may nolonger be sufficient to block differen-tiation and stimulate proliferationand as a consequence cellular dif-ferentiation increases. It should alsobe stressed that, although Fgf10 ap-pears to be crucial for pancreaticgrowth (Ohuchi et al., 2000; Bhushanet al., 2001), other factors, includingother FGFs and the EGF-family of sig-nalling factors, have also been sug-gested in pancreatic cell prolifera-tion (reviewed in Edlund, 2002),indicating that FGF10 may act inconcert with other factors to effec-tively stimulate pancreatic progeni-tor cell proliferation.

The reduced expression of Dll1and ngn3, together with the main-tained expression of Notch1 andHES-1 in the pancreatic epitheliumof Ipf1/Fgf10 mice, suggests that thestimulatory effect of Fgf10 on pan-creatic epithelial cell expansion, di-rectly or indirectly, involves Notchsignalling. Studies of neuronal differ-entiation in vitro are supportive of amechanism where FGFs stimulateproliferation and inhibit differentia-tion by means of the Notch pathway(Faux et al., 2001). The differentiationof neuroepithelial precursor cellscan be impaired by addition of ei-ther soluble Delta1 or FGFs (Faux etal., 2001). The addition of FGF1 or 2to neuroepithelial precursor cells re-sulted in an up-regulation of Notchexpression and a decrease in Delta1expression (Faux et al., 2001). Ourdata suggest that not only is theNotch-mediated lateral inhibitionmechanism conserved between theneuronal system and the pancreas(Lewis, 1996; Beatus and Lendahl,1998; Apelqvist et al., 1999; Grand-

wohl et al., 2000; Jensen et al., 2000)but also the potential role for FGFs asmoderators of Notch-signalling.

Genetic evidence from studies ofC. elegans sel-1 suggests that sel-1 isa negative regulator of Notch(Grant and Greenwald, 1997).Based on its homology to a gene inyeast, HRD3, which is required fordegradation of the HMG-CoA re-ductase, sel-1 has been proposed toregulate turnover of Notch, suggest-ing that absence of sel-1 may resultin an atypical accumulation of acti-vated Notch receptor (Grant andGreenwald, 1997). In the mousepancreas, Sel-1l expression starts tobe detectable from e13 and is highlyexpressed in the developing pan-creatic epithelium at this and laterstages of pancreatic developmentand in the adult pancreas (Donovielet al., 1998, Fig. 6I and data notshown). In the Ipf1/Fgf10 mice, Sel-1lexpression appears reduced, raisingthe possibility that continued highexpression of Fgf10 perturbs the initi-ation and/or level of Sel-1l expres-sion. If the role for Sel-1l in the devel-oping pancreas is to antagoniseactivated Notch, thus allowing celldifferentiation, then reduced Sel-1lexpression may result in an atypicalmaintenance of activated Notchthat would impair cell differentiation.The exact role for Sel-1l in relation toNotch activity and pancreas devel-opment need, however, to be eluci-dated through genetic manipula-tion of Sel-1l function in mice.

EXPERIMENTAL PROCEDURES

Generation of Ipf1/FGF10 Mice

A 4.5-kilobase (kb) NotI-NaeI frag-ment located immediately up-stream of the Ipf1/Pdx1 gene(Apelqvist et al., 1997) was sub-cloned into a vector carrying apolyA site and a 750-base pair (bp)NotI/NcoI fragment that corre-sponded to the full-length mouseFGF10 cDNA. Thus, spatial and tem-poral expression of the ligandthroughout development was en-sured. We generated transgenicmice by pronuclear injection of thepurified (NotI/BamHI; 1.8 ng/ml) intoF2 b6/CBA hybrid oocytes as de-scribed (Hogan et al., 1994). EightIpf1/Fgf10 founders were obtained

Fgf10 AND PANCREATIC EPITHELIAL CELLS 191

from 2 days of injections. Of theeight initial founders, one died at theneonatal stage, presenting the phe-notype described in the study. Fromthe resulting seven transgenicfounder mice, two failed to transmitthe transgene, whereas the remain-ing five (three males, two females),when bred with wild-type mice,transmitted the transgene to off-spring that presented with the phe-notypes described in the study.

Genotyping

The genotype of all offspring was de-termined by PCR analysis of genomicDNA extracted from tail biopsies orthe yolk sac by proteinase K (Boehr-inger) digestion and isopropanol pre-cipitation. The 5� and 3� primers used(amplified �500 bp) were 5�-TAGC-GAGGGGGAAGAGGAGAT-3� (Ipf1/Pdx-1 primer for 5�) and 5�-CTTA-CAGCTCCCAAGGGAATC-3� (theFGF10 primer for 3�). The PCR condi-tions used were as follows: 1 cycle of96°C for 5 min, 55°C for 2 min, and72°C for 3 min, followed by 29 cyclesof 94°C for 1 min, 55°C for 1 min,72°C for 3 min; and finally 1 cycle of72°C for 10 min.

In Situ Hybridization andImmunohistochemistry

In situ hybridization by using DIG-la-belled RNA probes for ngn3 (Apelqvistet al., 1997), Notch1 (Apelqvist et al.,1999), Dll1 (Apelqvist et al., 1999),HES-1 (Apelqvist et al., 1999), Sel-1L(Donoviel et al., 1998), and FGF10(Bellusci et al., 1997) was performedessentially as described (Ahlgren etal., 1996). Fgf10 in situ pictures in graymode were treated in false color byusing the single channel LUT display ofa NIKON EZ-C1 program (1.6 build 87,C1 hardware version 1.0, Nikon Cor-poration © 2002), and the incorporatescale was used to estimate the ex-pression levels. Immunohistochemicallocalization of antigens was per-formed as described (Ahlgren et al.,1996; Hart et al., 2000). The primaryantibodies used were rabbit anti-IPF1 (Ohlsson et al., 1993), rabbit an-ti-ISL1 (Thor et al., 1991), rabbit anti-FGFR2 (Research Diagnostics, Inc.),rabbit anti-phospho Histone H3 (Up-state Biotechnology), rabbit anti-

carboxypeptidase A (Anawa), rab-bit Nkx6.1 (raised against a GST-Nkx6.1 fusion protein by AgriSeraAB), rabbit anti-Nkx2.2 (Briscoe et al.,1999), rabbit anti-p48 (Li and Edlund,2001), mouse monoclonal anti-cyto-keratin 7 (DAKO), guinea pig anti-insulin (Linco), guinea pig anti-glu-cagon (Linco), rabbit anti-glucagon(Euro-Diagnostica), and rabbit anti-cleaved caspase 3 (Cell Signalling).The secondary antibodies usedwere fluorescein anti-guinea pig(Jackson), fluorescein anti-mouse(Jackson), and Cy3 anti-rabbit(Jackson). Quantification of totalpancreatic area was performed onthree independent transgenic e17pancreata by using a Zeiss Axion-plan 2 imaging microscope at amagnification of �5. The proliferat-ing cell cross-sectional area and to-tal pancreatic area were measuredby using the Image-Pro Plus 4.1 im-age analysis software (Media Cyber-netics). At least 40 sections, sepa-rated by 500 �m, were measuredper animal.

Confocal Microscopy

Longitudinal sections of dorsal pan-creata were immunostained as de-scribed above. Images were col-lected on a Leica TCS SP confocalmicroscope fitted with spectropho-tometer for emission band wave-length selection and dual detectorsand argon/krypton (Ar/Kr) andGre/Ne lasers for simultaneous scan-ning of two different fluorochromes.Confocal microscopy was used todetect the FGFR2 expression patternin wild-type. Sets of fluorescent im-ages were acquired simultaneouslyfor Cy3- and fluorescein-tagged an-ti-FGFR2 and anti-insulin or anti-glu-cagon markers, respectively. Com-panion images were scanned withpixel size 1 and 0.7-�m step size.Confocal image stacks were com-bined as x–y projection images, dig-itally optimized, and assigned redand green pseudocolours for Cy3and fluorescein, respectively.

Glucose Measurements

Blood samples were obtained fromthe tail vein from nonfasted animals(wild-type and transgenic) and glu-

cose levels were measured by usinga Precision Plus glucometer (Medi-sense).

RNA Extraction and ReverseTranscription

Total RNA was extracted from approx-imately 10–20 mg of pancreatic tis-sues from e15 embryos after homoge-nisation by using the NucleoSpin RNAII kit (BD Biosciences) following themanufacturer’s instructions. RNA wasreverse-transcribed by using the Su-perScript First-Strand Synthesis kit (In-vitrogen) again following the manu-facturer’s instructions.

Real Time PCR

Expression levels of fgf10 mRNA intransgenic (n � 3) and wild-typemice (n � 4) were determined byusing the ABI PRISM 7000 SequenceDetection System. PCR reactionswere carried out in 25-�l volumes in96-well plates, in a reaction buffercontaining 1� SYBR Green PCR Mas-ter Mix and 300 nmol of primers. Allreactions were run in duplicates witha preoptimised control primer pairfor �-actin, which enabled data tobe expressed in relation to an inter-nal reference, to allow for differ-ences in RT efficiency. Primers usedfor �-actin were 5�-GGCCAACCGT-GAAAAGATGA-3�and5�-ACGTAGC-CATCCAGGCTGTG-3�, for Fgf10were 5�-CAGCGGGACCAAGAATG-AAG-3� and 5�-TGACGGCAACAA-CTCCGATTT-3�. All primers annealedat 59°C and yielded amplicons of70–150 bp. Reaction conditionswere as follows: 95°C for 10 min, then40 cycles of 95°C for 15 sec, and60°C for 1 min. We quantified expres-sion levels by using the comparativeCt method. According to the man-ufacturer’s guidelines, data were ex-pressed as Ct values (the cycle num-ber where logarithmic PCR plotscross a calculated threshold line)and used to determine �Ct (�Ct �Ct of the target gene, e.g., Ct ofFgf10 minus the Ct of the house-keeping gene). Fold difference inexpression levels with relation to thereference gene was calculated byusing the equation 2-� �Ct (��Ct ��Ct Sample � �Ct calibrator). Mea-surements were carried out a mini-

192 HART ET AL.

mum of three times each andshowed a 1.5- to 2-fold increase inFgf10 expression in the transgeniccompared with wild-type mice.

ACKNOWLEDGMENTSWe thank Dr. Dorit Donoviel for theSel-1l cDNA, Dr. Thomas Edlund forcritical reading and comments, andDr. Stefan Norlin and other andmembers in our laboratory for help-ful discussions. H.E. received fundingfrom the Swedish Research Council;the Juvenile Diabetes Foundation,New York; the Wallenberg Consor-tium North; and the EU 5th programand EU regional fund, objective 1.

REFERENCES

Ahlgren U, Jonsson J, Edlund H. 1996. Themorphogenesis of the pancreatic mes-enchyme is uncoupled from that of thepancreatic epithelium in PDX1/IPF1-deficientmice.Development122:1409–1416.

Apelqvist A, Ahlgren U, Edlund H. 1997.Sonic hedgehog directs specialisedmesoderm differentiation in the intes-tine and pancreas. Curr Biol 7:801–804.

Apelqvist A, Li H, Sommer L. 1999. Notch-signalling controls pancreatic cell dif-ferentiation. Nature 400:877–881.

Beatus P, Lendahl U. 1998. Notch andneurogenesis. J Neurosci Res 54:125–136.

Bellusci S, Grindley J, Emoto H, Itoh N,Hogan B. 1997. Fibroblast growth factor10 (FGF10) and branching morpho-genesis in the embryonic mouse lung.Development 124:4867–4878.

Bhushan A, Itoh N, Kato S, Thiery JP, Cz-ernichow P, Bellusci S, Scharfmann R.2001. Fgf10 is essential for maintainingthe proliferative capacity of epithelialprogenitor cells during early pancre-atic organogenesis. Development 128:5109–5117.

Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, Rubenstein JL,Ericson J. 1999. Homeobox geneNkx2.2 and specification of neuronalidentity by graded Sonic hedgehogsignalling. Nature 398:622–627.

Celli G, LaRochelle WJ, Mackem S, SharpR, Merlino G. 1998. Soluble dominant-negative receptor uncovers essentialroles for fibroblast growth factors inmulti-organ induction and patterning.EMBO J 17:1642–1655.

Chiang MK, Melton DA. 2003. Single-celltranscript analysis of pancreas devel-opment. Dev Cell 4:383–393.

Donoviel DB, Donoviel MS, Fan E, Hadjan-tonakis A, Bernstein A. 1998. Cloning

and characterization of Sel-1l, a murinehomolog of the C. elegans sel-1 gene.Mech Dev 78:203–207.

Edlund H. 1998. Transcribing pancreas.Diabetes 47:1817–1823.

Edlund H. 2002. Pancreatic organogene-sis- developmental mechanisms andimplications for therapy. Nat RevGenet 3:524–532.

Elghazi L, Cras-Meneur C, Czernichow P,Scharfmann R. 2002. Role for FGFR2IIIb-mediated signals in controlling pancre-atic endocrine progenitor cell prolifer-ation. Proc Natl Acad Sci U S A 99:3884–3889.

Faux CH, Turnley AM, Epa R, Cappai R,Bartlett PF. 2001. Interactions betweenfibroblast growth factors and Notchregulate neuronal differentiation.J Neurosci 21:5587–5596.

Gradwohl G, Dierich A, LeMeur M, Guil-lemot F. 2000. Neurogenin3 is requiredfor the development of the four endo-crine cell lineages of the pancreas.Proc Natl Acad Sci U S A 9:71607–71611.

Grant B, Greenwald I. 1997. Structure,function, and expression of SEL-1, anegative regulator of LIN-12 and GLP-1in C. elegans. Development 124:637–644.

Hart AW, Baeza N, Apelqvist A, Edlund H.2000. Attenuation of FGF-signalling inmouse �-cells leads to diabetes. Na-ture 408:864–868.

Hogan B, Constantini F, Lacey E. 1994. Inmanipulating the mouse embryo: alaboratory manual. Cold Spring Har-bor, NY: Cold Spring Harbor Press.

Jensen J, Pedersen EE, Galante P. 2000.Control of endodermal endocrine de-velopment by Hes-1. Nat Genet 24:36–44.

Kato S, Sekine K. FGF-FGFR signaling invertebrate organogenesis. 1999. CellMol Biol 45:631–638.

Kawaguchi Y, Cooper B, Gannon M, RayM, MacDonald RJ, Wright CV. 2002.The role of the transcriptional regulatorPtf1a in converting intestinal to pan-creatic progenitors. Nat Genet 32:128–134.

Krapp A, Knofler M, Ledermann B, Burki K,Berney C, Zoerkler N, Hagenbuchle O,Wellauer PK. 1998. The bHLH proteinPTF1-p48 is essential for the formation ofthe exocrine and the correct spatialorganization of the endocrine pan-creas. Genes Dev 12:3752–3763.

Lewis J. 1996. Neurogenic genes and ver-tebrate neurogenesis. Curr Opin Neu-robiol 6:3–10.

Li H, Edlund H. 2001. Persistent expressionof Hlxb9 in the pancreatic epitheliumimpairs pancreatic development. DevBiol 240:247–253.

Miralles F, Czernichow P, Ozaki K, Itoh N,Scharfmann R. 1999. Signaling throughfibroblast growth factor receptor 2bplays a key role in the development of

the exocrine pancreas. Proc Natl AcadSci U S A 96:6267–6272.

Ohlsson H, Karlsson K, Edlund T. 1993. IPF1,a homeodomain-containing transacti-vator of the insulin gene. EMBO J 12:4251–4259.

Ohuchi H, Hori Y, Yamasaki M, Harada H,Sekine K, Kato S, Itoh N. 2000. FGF10acts as a major ligand for FGF receptor2 IIIb in mouse multi-organ develop-ment. Biochem Biophys Res Commun277:643–649.

Revest JM, Spencer-Dene B, Kerr K, DeMoerlooze L, Rosewell I, Dickson C.2001. Fibroblast growth factor receptor2-IIIb acts upstream of Shh and Fgf4and is required for limb bud mainte-nance but not for the induction of Fgf8,Fgf10, Msx1, or Bmp4. Dev Biol 231:47–62.

Ryan EA, Lakey JR, Rajotte RV, KorbuttGS, Kin T, Imes S, Rabinovitch A, ElliottJF, Bigam D, Kneteman NM, WarnockGL, Larsen I, Shapiro AM. 2001. Clinicaloutcomes and insulin secretion after is-let transplantation with the Edmontonprotocol. Diabetes 50:710–719.

Sander M, Sussel L, Conners J, Scheel D,Kalamaras J, Dela Cruz F, SchwitzgebelV, Hayes-Jordan A, German M. 2000.Homeobox gene Nkx6.1 lies down-stream of Nkx2.2 in the major pathwayof beta-cell formation in the pancreas.Development 127:5533–5540.

Schwitzgebel VM, Scheel DW, ConnersJR, Kalamaras J, Lee JE, Anderson DJ,Sussel L, Johnson JD, German MS. 2000.Expression of neurogenin3 reveals anislet cell precursor population in thepancreas. Development 127:3533–3542.

Selander L, Edlund H. 2002. Nestin is ex-pressed in mesenchymal and not epi-thelial cells of the developing mousepancreas. Mech Dev 113:189–192.

Shapiro AM, Lakey JR, Ryan EA, KorbuttGS, Toth E, Warnock GL, KnetemanNM, Rajotte RV. 2000. Islet transplanta-tion in seven patients with type 1 dia-betes mellitus using a glucocorticoid-free immunosuppressive regimen.N Engl J Med 343:230–238.

Sussel L, Kalamaras J, Hartigan-O’ConnorDJ, Meneses JJ, Pedersen RA, Ruben-stein JL, German MS. 1998. Mice lack-ing the homeodomain transcriptionfactor Nkx2.2 have diabetes due to ar-rested differentiation of pancreaticbeta cells. Development 125:2213–2221.

Szebenyi G, Fallon JF. 1999. Fibroblastgrowth factors as multifunctional sig-naling factors. Int Rev Cytol 185:45–106.

Thor S, Ericson J, Brannstrom T, Edlund T.1991. The homeodomain LIM proteinIsl-1 is expressed in subsets of neuronsand endocrine cells in the adult rat.Neuron 7:881–889.

Fgf10 AND PANCREATIC EPITHELIAL CELLS 193