Rbp-j regulates expansion of pancreatic epithelial cells and their differentiation into exocrine...

RESEARCH ARTICLE

Rbp-j Regulates Expansion of PancreaticEpithelial Cells and Their Differentiation IntoExocrine Cells During Mouse DevelopmentJunji Fujikura,1 Kiminori Hosoda,1* Yoshiya Kawaguchi,2 Michio Noguchi,1 Hiroshi Iwakura,1

Shinji Odori,1 Eisaku Mori,1 Tsutomu Tomita,1 Masakazu Hirata,1 Ken Ebihara,1 Hiroaki Masuzaki,1

Akihisa Fukuda,2,3 Kenichiro Furuyama,2 Kenji Tanigaki,4 Daisuke Yabe,5 and Kazuwa Nakao1

Notch signaling regulates cell fate determination in various tissues. We have reported the generation ofmice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited prematureendocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated inPtf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-jf/f Ptf1a.cremice is 1 day later than that in Rbp-jf/f Pdx.cre mice. In Rbp-jf/f Ptf1a.cre mouse pancreases, at E13.5, thereduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine celldifferentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation wasreduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our studyindicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-jregulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreaticdevelopment. Developmental Dynamics 236:2779–2791, 2007. © 2007 Wiley-Liss, Inc.

Key words: pancreatic development; Rbp-j; Notch signaling; Pdx1; Ptf1a; conditional knockout (KO) mice

Accepted 26 July 2007

INTRODUCTION

The pancreas plays a key role in themaintenance of nutritional homeosta-sis through its exocrine and endocrinefunctions. The exocrine acini secretedigestive enzymes into the duodenumthrough ducts, whereas the endocrinecells in the islets produce peptide hor-mones (Heller et al., 2005). Notch sig-naling regulates various developmen-tal processes, such as neurogenesis,

somitogenesis, angiogenesis, and he-matopoiesis (Ishibashi et al., 1995;Hrabe de Angelis et al., 1997; Xue etal., 1999; Han et al., 2002). The inter-action of a Notch receptor with its li-gand induces the cleavage of the re-ceptor’s intracellular domain (NotchICD), which translocates to the nu-cleus and binds to Rbp-j to induce theexpression of the Hes family of tran-scriptional repressors (Kageyama and

Ohtsuka, 1999). Rbp-j is a key media-tor of Notch signaling, because it isexpressed ubiquitously and associateswith all four types of Notch receptors(Kato et al., 1996; Beres et al., 2006).Various Notch-related genes are ex-pressed in the developing pancreas(Lammert et al., 2000). However, mul-tiple anomalies and early embryonicdeaths of mice with homozygous dele-tions of such genes limit the possible

The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat1Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan2Department of Surgery and Surgical Basic Science, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan3Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan4Research Institute, Shiga Medical Center, Moriyama, Shiga, Japan5Department of Medical Chemistry, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, JapanGrant sponsor: the Ministry of Education, Culture, Sports, Science, and Technology of Japan; Grant number: Grant-in-aid; Grant sponsor:The Smoking Research Foundation.*Correspondence to: Kiminori Hosoda, Department of Medicine and Clinical Science, Kyoto University Graduate School ofMedicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: [email protected]

DOI 10.1002/dvdy.21310Published online 7 September 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:2779–2791, 2007

© 2007 Wiley-Liss, Inc.

assessment of the importance of Notchsignaling in this organ (Swiatek et al.,1994; Ishibashi et al., 1995; Oka et al.,1995; Hrabe de Angelis et al., 1997;Xue et al., 1999; Apelqvist et al., 1999;Hamada et al., 1999; Jensen et al.,2000). We recently reported the gen-eration of mice in which the Rbp-jgene is inactivated by Cre recombi-nase under the control of the Pdx1promoter (Rbp-jf/f Pdx.cre mice; Fu-jikura et al., 2006). Pdx1 is a ho-meobox transcription factor requiredfor pancreatic outgrowth and the dif-ferentiation of the rostral duodenum(Offield et al., 1996). This protein isfirst detected in the endodermal do-mains of the foregut–midgut junctionfrom as early as embryonic day (E) 8.5(Guz et al., 1995; Offield et al., 1996;Edlund, 2002). In Rbp-jf/f Pdx.cremice, the premature differentiation ofpancreatic progenitor cells into endo-crine and ductal cells leads to pancre-atic hypoplasia and insulin-deficientdiabetes mellitus. Ptf1a, like Pdx1, isa transcription factor that is ex-pressed in pancreatic progenitors ofexocrine, endocrine, and duct cellswithin the pancreatic buds of the earlyembryo. Ptf1a is a basic helix–loop–helix type of transcription factor,which is necessary for pancreaticspecification and early organogenesis(Cockell et al., 1989; Kawaguchi et al.,2002). Ptf1a protein can be found atE10.5, shortly after the onset ofendodermal budding, only in the cellsthat become committed to pancreaticfates (Hald et al., 2003). In this study,to further elucidate the role of Rbp-j–mediated Notch signaling in the de-veloping pancreas using the differenttimes of expression of Ptf1a and Pdx1,we disrupted Rbp-j with Cre recombi-nase under the control of the endoge-nous Ptf1a promoter. In Rbp-jf/f

Ptf1a.cre mouse pancreases, the num-ber of Hes1-positive cells decreased,but premature endocrine or ductaldifferentiation was minimal. Thebranching morphogenesis of the epi-thelium and acinar cell differentiationwere disturbed with reduced Pdx1 ex-pression in the mutant pancreases.Our study indicates that Rbp-j regu-lates epithelial growth, Pdx1 expres-sion, and acinar cell differentiationduring mid-pancreatic development.

RESULTS

Timing of Cre/loxPRecombination in Rbp-jf/f

Ptf1a.cre Mice IsDevelopmentally Later ThanThat in Rbp-jf/f Pdx.cre Mice

To study the role of Rbp-j in pancreaticdevelopment and function, we condi-tionally deleted the gene for Rbp-j in thedeveloping pancreas. In the floxed alleleof Rbp-j, loxP sites were introduced up-stream from exon 6 and downstreamfrom exon 7 of the gene (Han et al.,2002). Exons 6 and 7 encode DNA-bind-ing and Notch-binding domains, andthe lack of these exons results in thecomplete loss of Rbp-j–mediated Notchsignaling. Rbp-jf is a very efficient tar-get for Cre recombinase in multiple tis-sues (Han et al., 2002; Tanigaki et al.,2002, 2004; Yamamoto et al., 2003; Fu-jikura et al., 2006; Buono et al., 2006).Recently, we reported the generation ofmice in which the Rbp-j gene was inac-tivated by Cre recombinase under thecontrol of the Pdx1 promoter (Rbp-jf/f

Pdx.cre; Fujikura et al., 2006). We nextmated Rbp-jf/f mice with mice express-ing Cre recombinase driven by the en-dogenous Ptf1a promoter (Rbp-jf/f

Ptf1a.cre; Kawaguchi et al., 2002). Wemonitored the timing of Cre/loxP re-combination using Rosa26-lacZ re-porter (Rosa26R) mice. The Rosa26Rallele carries a histochemical markerlacZ gene driven by the promoter ofthe ubiquitously expressed Rosa26gene in a configuration such that tran-scription is terminated upstream fromthe lacZ reporter by floxed stop se-quences (Soriano, 1999). To visualizethe Cre-mediated loxP recombination,mice heterozygous for floxed Rbp-jand homozygous for the Rosa26R al-lele, Rosa26Rf/f Rbp-jf/� were crossedwith double heterozygous Rbp-jf/�

Ptf1a.cre or Rbp-jf/� Pdx.cre mice. Inthe offspring, the expression of Crerecombinase excises both the stop cas-sette upstream from the lacZ gene andthe genomic region from exons 6 to 7of the Rbp-j gene, thereby activatingthe expression of �-galactosidase andinactivating that of Rbp-j. Because re-combination is never reversed, lacZexpression persists in all descendantsof the cells in which the Rbp-j gene isdeleted from the genome. X-gal (5-Bromo-4-chloro-3-indolyl-�-D-galacto-

side) staining of Rosa26Rf/� Rbp-jf/f

Pdx.cre embryos appeared betweensomite stages 19 and 22 (E9.5) inthe dorsal and ventral pancreaticbuds of the foregut region (Fig. 1, up-per panels). However, in theRosa26Rf/� Rbp-jf/f Ptf1a.cre embryos,X-gal staining first appeared somitestage 32 or 33 (E10.5) in the dorsal andventral pancreatic buds (Fig. 1, lowerpanels). Thus, the timing of the genedeletion in the Rbp-jf/f Ptf1a.cre micewas approximately 1 day later develop-mentally than that in the Rbp-jf/f Pdx-.cre mice. At various developmentalstages in Rosa26Rf/� Rbp-jf/� Ptf1a.creand Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice,we generally detected uniform �-galac-tosidase activity throughout the pancre-atic tissues (Supplementary FigureS1A–1E, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat), but not in earlyglucagon-producing cells (Supple-mentary Figure S1C).

A paralog of Rbp-j, Rbp-L is ex-pressed in pancreatic acinar cells (Mi-noguchi et al., 1997; Beres et al.,2006). The Rbp-L protein binds to aDNA sequence almost identical tothat of Rbp-j, but does not interactwith any of the four known mamma-lian Notch receptors (Minoguchi et al.,1997). No compensatory up-regulationof Rbp-L could be detected by immu-nostaining with specific antibody(Supplementary Figure S2).

Decreased Epithelial CellProliferation and ReducedBranching Morphogenesis inthe Pancreases of Rbp-jf/f

Ptf1a.cre Mice at E13.5

At E13.5, the Pdx1-positive pancreaticepithelium of the control mice had al-ready started to branch (Fig. 2Aa).However, in Rbp-jf/f Ptf1a.cre mice,the Pdx1-positive epithelium rarelybranched, and dilated tubular struc-tures were prominent (Fig. 2Ab). Inthe control pancreas, Hes1 expressionwas broadly distributed in the branch-ing epithelium (Fig. 2Ac) and neuro-genin 3 expression was mainly de-tected in cells located in the centralregion of the epithelium (Fig. 2Ae). Inthe mutant pancreas, the ratio ofHes1-positive cells to Pdx1-positivecells (Hes1�/Pdx1�) significantly de-

2780 FUJIKURA ET AL.

creased to 55% of the control (Rbp-jf/�

Ptf1a.cre, 24.1 � 2.5 vs. Rbp-jf/f

Ptf1a.cre, 13.3 � 1.7%; P � 0.01; Fig.2Ac,Ad,B), and neurogenin 3�/Pdx1�ratio increased to 112% of the control,but was not significant (Rbp-jf/�

Ptf1a.cre, 5.1 � 0.4 vs. Rbp-jf/f

Ptf1a.cre, 5.8 � 0.8%; P � 0.42; Fig.2Ae,Af,B). Most of the cells in the di-

lated tubular structures stained neg-atively for both Hes1 (Fig. 2Ad) andneurogenin 3 (Fig. 2Af). Synaptophy-sin is a marker for neuroendocrinecells (Wiedenmann et al., 1986). Syn-aptophysin-positive cell clusters wereobserved in both the control and mu-tant pancreases (Fig. 2Ag,Ah). Lin-eage tracing experiments showed that

these large clusters were mainly com-posed of glucagon-expressing cells re-taining unrecombined allele (Supple-mentary Figure S1C). However,synaptophysin�/ Pdx1� ratio in-creased to 186% of the control (Rbp-jf/� Ptf1a.cre, 5.2 � 1.0 vs. Rbp-jf/f

Ptf1a.cre, 9.8 � 1.6%; P � 0.04; Fig.2Ag,Ah,B), reflecting to some extent thedecreased number of Pdx1-positive cells(Fig. 2Ab). The expression of car-boxypeptidase A, an earlier exocrinecell marker than amylase (Gittes andRutter, 1992), was detected peripher-ally at the tip of the branches in thecontrol pancreas (Fig. 2Ai). In contrast,carboxypeptidase-A–expressing cellswere rarely found in the mutant (Fig.2Aj). Reduced outgrowth and branchingof the epithelium in the mutant wasaccompanied by a decrease in the num-ber of proliferating cells detected byphospho-histone H3 immunostaining(Fig. 2Ak,Al). The phospho-histoneH3�/ Pdx1� ratio decreased to 40% ofthe control (Rbp-jf/� Ptf1a.cre, 4.4 � 0.6vs. Rbp-jf/f Ptf1a.cre, 1.8 � 0.5%; P �

Fig. 1. Timing of Cre/loxP recombination in Rbp-jf/f Ptf1a.cre mice is later developmentally thanin Rbp-jf/f Pdx.cre mice. X-gal staining of whole embryos from Rosa26Rf/� Rbp-jf/f Pdx.cre mice(upper panels) and Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice (lower panels) at embryonic days (E) 8.5,E9.5, E10.5, and E11.5. Pdx.cre-mediated recombination is first detected at E9.5, but Ptf1a.cre-mediated recombination is first detected at E10.5. Scale bars � 200 �m.

Fig. 2. Decreased epithelial cell proliferation and reduced branching morphogenesis in the pancreases of Rbp-jf/f Ptf1a.cre mice at embryonic day(E) 13.5. A: Immunohistochemistry of Pdx1 (a,b), Hes1 (c,d), neurogenin 3 (e,f), synaptophysin (g,h), carboxypeptidase A (i,j), phospho-histone H3 (k,l),Dolichos biflorus agglutinin (DBA; m,n) using serial pancreatic sections from Rbp-jf/� Ptf1a.cre and Rbp-jf/f Ptf1a.cre mice at E13.5. B: The relativenumbers of Hes1, neurogenin 3, synaptophysin, and phospho-histone H3–positive cells to Pdx1-positive cells. Standard errors and number of miceexamined are indicated on the average bars. Levels of significance (Student’s t-tests) are shown. In the mutant pancreas, epithelial growth andbranching is significantly reduced. Scale bars � 50 �m.

RBP-J AND MOUSE PANCREATIC EPITHELIAL CELLS 2781

Fig. 3.

Fig. 4.


0.01; Fig. 2B). No cleaved caspase-3staining was observed in either the con-trol or the mutant pancreases (data notshown). In the mutant pancreas, partsof the cells lining the lumens of dilatedtubular structures were positivelystained with Dolichos biflorus aggluti-nin (DBA; Fig. 2An).

Reduced Pdx1 Expressionand Poor Exocrine CellDifferentiation in thePancreases of Rbp-jf/f

Ptf1a.cre Mice at E15.5

At E15.5, the Pdx1-positive pancreaticepithelium of the control mice exhibitedcomplex and ramified networks (Fig.3Aa,Ba). However, in Rbp-jf/f Ptf1a.cremice, the branching of the epitheliumwas reduced, and dilated tubular struc-tures remained (Fig. 3Ab). The epithe-lial cells, especially in the dilated tubu-lar walls, were negative or faintlystained for Pdx1 (Fig. 3Ab,Bb). Ataround this stage, Hes1 expression wasdiffusely detected in the pancreaticmesenchyme (Fig. 3Ac,Ad). In the con-trol epithelium, Hes1-positive cellswere reduced compared with those atearlier stages, but were still present(Fig. 3Ac), and neurogenin 3 expressionpeaked between E13.5 and E15.5 (Figs.2e, 3Ae). In contrast, Hes1 expressionwas significantly decreased in the mu-tant epithelium (Fig. 3Ad) and the mostof the cells of the dilated tubular struc-tures remained negative for both Hes1(Fig. 3Ad) and neurogenin 3 (Fig. 3Af).

The number of endocrine cells per sec-tion detected by synaptophysin (Fig.3Ag,Ah), glucagon (Fig. 3Ai,Aj), and in-sulin (Fig. 3Ak,Al) immunostaining didnot seem to change substantially. Weestimated the total endocrine cell massfrom the synaptophysin-positive area inmultiple, evenly spaced sections takenthrough the pancreas. In the mutantpancreas, the endocrine cell mass de-creased to 87% of that of the control(Rbp-jf/� Ptf1a.cre, 205,736 � 7,194 vs.Rbp-jf/f Ptf1a.cre, 178,745 � 3,954 pix-els; P � 0.02). At this stage, amylase-positive cells were present in the acinarregion of the control pancreas (Fig.3Am), but these cells were significantlyreduced in number in the mutant pan-creas (Fig. 3An). Most of the cells liningthe lumens of dilated tubular structuresin the mutant pancreas stained posi-tively with DBA (Fig. 3Ap), and thenumber of phospho-histone H3–positiveproliferating cells was reduced (Fig. 3Arcompared with Fig. 3Aq). We next usedimmunofluorescence to further investi-gate the expression and localization ofPdx1, amylase, and phospho-histoneH3. Pdx1 was uniformly expressed inthe control pancreas, and all amylase-expressing cells also stained for Pdx1(Fig. 3Ba,Bc,Be). However, in the mu-tant, Pdx1 expression was significantlyreduced (Fig. 3Bb) and exocrine cell dif-ferentiation was severely impaired (Fig.3Bd). In the control pancreas, differen-tiated exocrine cells exhibited active di-vision (Fig. 3Bg). However, both exo-crine cells and proliferating cells were

sparsely distributed in the mutant pan-creas (Fig. 3Ar,An,Bh). No cleavedcaspase-3 staining was observed in boththe control and the mutant pancreas(data not shown).

Small Pancreas WithIncreased Exocrine CellProliferation in Rbp-jf/f

Ptf1a.cre Mice at PostnatalDay 0

At postnatal day (P) 0, total pancreaticsize was obviously decreased (Fig. 4A),but islet formation was more prominentin the Rbp-jf/f Ptf1a.cre mice than in thecontrol mice (Fig. 4Ba–Bh). Cytokera-tin-positive dilated ductular structuresoccupied a small part of the mutantpancreas (Fig. 4Bj). The area of amy-lase-positive cells was significantlysmaller in the mutant pancreas (Fig.4Bl) than that in the control pancreas(Fig. 4Bk). Pdx1 expression was barelydetectable in the exocrine cells of boththe control and mutant pancreases atthis time (Fig. 4Ca,Cb). The prolifera-tion rate of the exocrine cells was muchlower than at E13.5 in the control pan-creas (Fig. 4Cc compared with Fig.3Bg). However, the exocrine cells of themutant pancreas showed active prolif-eration, as if in compensation for inad-equate differentiation (Fig. 4A, Cd). Thepercentage of proliferating acinar cellswas significantly increased comparedwith the control group (Rbp-jf/f, 0.16 �0.01 vs. Rbp-jf/f Ptf1a.cre, 2.03 � 0.15%;P � 0.01).

We traced the lineage of Rbp-j–de-leted pancreatic cells, because the his-tological changes in the mutant pan-creas were not simple or uniform.�-Galactosidase activity was observedbroadly and uniformly throughout thepancreatic tissues, including endo-crine, ductal, and exocrine cells, in theRosa26Rf/� Rbp-jf/f Ptf1a.cre mice(Supplementary Figure S1E).

Catch-Up Growth ofExocrine Tissue in thePancreases of Rbp-jf/f

Ptf1a.cre Mice During theFirst 3 Postnatal Weeks

Significant catch-up growth occurredduring the early postnatal weeks inthe exocrine tissue of the Rbp-jf/f

Fig. 3. Reduced Pdx1 expression and poor exocrine cell differentiation in pancreas of Rbp-jf/f

Ptf1a.cre mice at embryonic day (E) 15.5. A: Immunohistochemistry of Pdx1 (a,b), Hes1 (c,d),neurogenin 3 (e,f), synaptophysin (g,h), glucagon (i,j), insulin (k,l), amylase (m,n), Dolichos biflorusagglutinin (DBA; o,p), and phospho-histone H3 (q,r) in the pancreas of Rbp-jf/� Ptf1a.cre andRbp-jf/f Ptf1a.cre mice at E15.5. Except for DBA (o,p) and phospho-histone H3 (q,r), serial sectionsare stained. The framed areas in i and k, or j and l indicate continuous areas of the samegenotypes. In the mutant pancreas, the areas of Pdx1-positive (b), Hes1-positive (d), and neuro-genin 3-positive (f) cells are limited and the acinar formation is poor. B: Double immunofluores-cence against Pdx1 (green; a,b) and amylase (red; c,d) and their merged images (e,f), and mergedimages of double immunofluorescence against phospho-histone H3 (green) and amylase (red;g,h). The number of Pdx1/Amylase co-positive cells and proliferating cells are significantlydecreased in the mutant pancreas. Scale bars � 50 �m in A, 100 �m in B.

Fig. 4. Small pancreas with increased exocrine cell proliferation in Rbp-jf/f Ptf1a.cre mice atpostnatal day (P) 0. A: Macroscopic dorsal view of the X-gal stained pancreases from Rosa26Rf/�

Rbp-jf/� Ptf1a.cre mice (left) and Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice (right) at P0. B: Immunohis-tochemical studies with antibodies to insulin (a,b), glucagon (c,d), somatostatin (e,f), pancreaticpolypeptide (g,h), cytokeratin (i,j), and amylase (k,l) using serial pancreatic sections from Rbp-jf/�

Ptf1a.cre and Rbp-jf/f Ptf1a.cre mice at P0. Islet formation is more prominent in the mutantpancreas. C: Merged images of double immunofluorescences against Pdx1 (green) and amylase(red) (a,b) or phospho-histone H3 (green) and amylase (red; c,d). The proportion of proliferatingamylase-positive cells is 13-fold higher in the mutant. Scale bars � 100 �m.


Ptf1a.cre mice. At postnatal day 14,amylase-positive cells were distrib-uted patchily in the mutant comparedwith those in the control (Fig. 5A).However, at postnatal day 21, the mu-tant pancreas showed apparently nor-mal histology and morphology, indis-tinguishable from that of the controlpancreas (Fig. 5B,C).

Normal Pancreas, Except forSmall Ductal Remnants, inAdult Rbp-jf/f Ptf1a.cre Mice

The adult Rbp-jf/f Ptf1a.cre mice hadovertly normal pancreases (Fig. 6A).No mosaic pattern of recombinationwas apparent by X-gal staining of pan-creatic sections (Fig. 6B). The deletion

efficiency was assessed by Southernblot analysis of SphI-digested genomicDNA prepared from adult pancreatictissues (Fig. 6C). Only the unrecom-bined floxed allele was detected in the1.2-kb band from the tail tissue, andthe recombined null allele was de-tected in the 3-kb band from the pan-creas and the pool of islets. We found

Fig. 5. By postnatal day (P) 21, Rbp-j–deficient acinar tissue showed apparently normal morphology indistinguishable from control. A,B: Immuno-histochemical studies with antibodies to insulin, glucagon, cytokeratin, amylase, and phospho-histone H3 using serial pancreatic sections fromRbp-jf/� Ptf1a.cre and Rbp-jf/f Ptf1a.cre mice at P14 (A), and at P21 (B). C: X-gal staining of pancreatic regions from Rosa26Rf/� Rbp-jf/f mice (left) andRosa26Rf/� Rbp-jf/f Ptf1a.cre mice (right) at P21. Scale bars � 200 �m.


almost complete and ubiquitous re-combination of the floxed allele in theadult pancreases of the Rbp-jf/f

Ptf1a.cre mice. The ratio of pancreaticweight to total body weight (Rbp-jf/f,0.90 � 0.09 vs. Rbp-jf/f Ptf1a.cre,0.71 � 0.10%; P � 0.28; Fig. 6D) wasnot altered in the mutants comparedwith that of their control littermates.The endocrine cell mass was quanti-fied by estimating the hormone-posi-tive area per total pancreatic area inmultiple pancreatic sections. The rel-ative cell areas of the � and � cell werenot significantly altered in the Rbp-jf/f

Ptf1a.cre mice (relative � cell area:Rbp-jf/f, 1.24 � 0.25 vs. Rbp-jf/f

Ptf1a.cre, 1.31 � 0.30%; P � 0.85; rel-ative � cell area: Rbp-jf/f, 0.19 � 0.03mg/dl vs. Rbp-jf/f Ptf1a.cre, 0.26 �0.04%; P � 0.20; Fig. 6E). Hematoxy-lin-eosin staining and immunohisto-chemical studies with antibodies di-rected against glucagon, insulin, Pdx1,somatostatin, pancreatic polypeptide,cytokeratin, and amylase revealed noabnormalities in the islet structures(Fig. 6F). In our previously reportedRbp-jf/f Pdx.cre mice, the pancreasshowed striking changes in the pancre-atic cell composition of the adult. Cytok-eratin-positive ductal cells were abun-dantly present, whereas there werenoticeable cystic ductal dilations (Fig.7A, middle panels). However, in theRbp-jf/f Ptf1a.cre mice at the same age,we observed only a focal area of ductalstructures at the tip of the pancreas(Fig. 7A, lower panels). We confirmedby lineage tracing that all of these duc-tal and ductular structures were alsoderived from Rbp-j–deficient cells (Fig.7B, blue staining).

No Signs of Endocrine orExocrine PancreaticInsufficiency in Rbp-jf/f

Ptf1a.cre Mice

Rbp-jf/f Ptf1a.cre mice had normalbody weights (Rbp-jf/f, 27.9 � 1.6 g vs.Rbp-jf/f Ptf1a.cre, 30.2 � 1.5 g; P �0.35; Fig. 8A). No significant differ-ences were detected in the levels ofblood glucose (Rbp-jf/f, 117 � 8 mg/dlvs. Rbp-jf/f Ptf1a.cre, 119 � 3 mg/dl;P � 0.76; Fig. 8B) or plasma insulin(Rbp-jf/f, 2.08 � 0.34 ng/ml vs. Rbp-jf/f

Ptf1a.cre, 2.07 � 0.40 ng/ml; P � 0.99;Fig. 8C) in the fed animals. Duringintraperitoneal glucose tolerance test-

ing (2 g/kg body weight), the Rbp-jf/f

Ptf1a.cre mice cleared excess bloodglucose at a rate similar to that of theRbp-jf/f mice (Fig. 8D). Serum amylaselevels were not significantly altered inthe Rbp-jf/f Ptf1a.cre mice comparedwith the control Rbp-jf/f mice (Rbp-jf/f,1,214 � 26 U/dl vs. Rbp-jf/f Ptf1a.cre,1,291 � 102 U/dl; P � 0.41; Fig. 8E).

DISCUSSION

Using stage-specific conditional genetargeting, we documented the effectsof Rbp-j–mediated Notch signaling onpancreatic development and function.We recently reported the generation oftwo types of conditional Rbp-j knock-out mice (Fujikura et al., 2006). Rbp-jf/f Pdx.cre mice, in which Rbp-j wasinactivated using a Pdx.cre transgene,exhibited insulin-deficient diabeteswith small pancreases, few islets, andlower insulin content. Pancreatic hyp-oplasia was caused by the prematuredepletion of progenitor cells and theprecocious differentiation of endocrineand ductal cells. Rbp-jf/f Rip.cre mice,in which the Rbp-j gene was inacti-vated using a Rip.cre transgene,showed normal function and prolifer-ation of the pancreatic � cells. To fur-ther assess the effects of Rbp-j–medi-ated Notch signaling on pancreaticdevelopment, we disrupted Rbp-j withCre recombinase under the control ofthe endogenous Ptf1a promoter. Weused recombination at the Rosa26Rallele as a surrogate marker for therecombination-induced deletion ofRbp-j, because there are no estab-lished methods for the histological de-tection of Rbp-j. It is reasonable toassume that the difference in onset ofX-gal staining between Ptf1a.cre andPdx.cre driver lines reflects the onsetof gene expression between Ptf1a andPdx1 (Guz et al., 1995; Offield et al.,1996; Edlund, 2002; Li and Edlund,2002). Therefore, we consider that thetiming of the Rbp-j deletion in pan-creas development in Rbp-jf/f Ptf1a.cremice is approximately 1 day later thanin Rbp-jf/f Pdx.cre mice. However, spe-cific and sensitive methods to directlydetect and quantify Rbp-j protein insitu would be needed to confirm ourresults, because recombination at aRosa26 allele does not always guaran-tee deletion of the floxed Rbp-j loci.The difference in timing critically in-

fluenced the phenotypes of the condi-tional Rbp-j knockout mice, becauseRbp-jf/f Ptf1a.cre mice had minimalsigns of premature endocrine and duc-tal differentiation and showed suffi-cient pancreatic functions at adultages. These results narrow the timewindow when Notch/Rbp-j signalingplays a critical role in the develop-ment and homeostasis of the pan-creas.

In the pancreatic buds from bothRbp-jf/f Pdx.cre mice and Rbp-jf/f

Ptf1a.cre mice, not all the cells lostHes1 expression with the loss of Rbp-j.This observation suggests that thepancreatic buds contain differentclasses of progenitors with respect totheir sensitivity to Rbp-j–mediatedNotch signaling and that some otherfactors are also involved in maintain-ing Hes1 expression in the epithelium.Recently, Sox9 was shown to be one ofsuch factors (Seymour et al., 2007).

The forced expression of the consti-tutively active form of the Notch1 re-ceptor (Notch1 ICD) in the early pan-creatic progenitors traps them in anundifferentiated state (Hald et al.,2003; Esni et al., 2004). Using tamox-ifen-inducible expression of Notch1ICD in mice, Murtaugh et al. alsodemonstrated that Pdx1-positive pro-genitors are sensitive to Notch signal-ing, even if Notch activation occurslong after the pancreas has been spec-ified (Murtaugh et al., 2003). Funda-mentally, Rbp-jf/f Pdx.cre mice havethe reverse phenotype, with prema-ture endocrine and ductal differentia-tion (Fujikura et al., 2006). However,our study indicates that it is in thevery early stages of development thatNotch signaling plays a critical rolefor the pancreas. This differencemight have arisen from the differentexperimental conditions used. Onestudy used gene disruption (loss-of-function) and the other used overex-pression (gain-of-function) experi-ments. Alternatively, it may havebeen caused by differences in themodes of actions of Rbp-j and theNotch ICD.

We detected defective branchingmorphogenesis in both Rbp-jf/f Pdx.creand Rbp-jf/f Ptf1a.cre mice at aroundE13.5. Branching morphogenesis, de-fined as the growth and branching ofepithelial tubules during embryogen-esis, is a fundamental feature of renal,


lung, mammary gland, submandibu-lar gland, and pancreatic morphogen-esis in mammals. In embryos, thepancreatic branching structures con-tain undifferentiated precursor cells,which migrate away to generate isletand acinar cells (Pictet and Rutter,1972; Teitelman et al., 1993). A seriesof studies revealed that, apart from itswell-documented involvement in cellfate decisions, Notch signaling couldaffect cellular proliferation (Deftos etal., 1998; Shelly et al., 1999; Satoh etal., 2004). During prostate develop-

Fig. 6.

Fig. 7.


ment, Notch1-expressing cells definedthe progenitor cells in the prostaticepithelial cell lineage, and ablationof Notch signaling in those cells in-hibits the branching morphogenesis,growth, and differentiation (Wanget al., 2004). In the pancreas, dis-turbed branching morphogenesisalso occurs in mice deficient in Fgf10

or its high-affinity receptor Fgfr2-IIIb (Bhushan et al., 2001; Pulkki-nen et al., 2003). Furthermore, theoverexpression of Fgf10 in the pan-creatic epithelium causes increasedproliferation and the persistent up-regulation of Notch components inthe epithelium (Norgaard et al.,2003; Hart et al., 2003). It has re-

cently been reported that Hes1 pro-motes epithelial cell proliferation byrepressing the expression of thecyclin-dependent kinase inhibitorp57Kip2 during the early stages ofpancreatic development (Georgia etal., 2006). Taking these data to-gether, we consider that active Rbp-j–mediated Notch signaling cell-au-tonomously helps to maintain a poolof epithelial progenitors due, at leastin part, to a Hes1-mediated path-way.

Both Hes1 and Pdx1 are expressedin the initial stage of exocrine cell dif-ferentiation (Esni et al., 2004). InRbp-jf/f Ptf1a.cre mice, the lack ofRbp-j results in the reduction of bothHes1 and Pdx1 protein expression, ac-companied by a decrease in acinar celldifferentiation at E15.5. Hale et al.showed that the depletion of Pdx1 atE12.5 prevents the expression of Ptf1aand halts epithelial differentiationjust before the formation of acini(Hale et al., 2005). Ptf1a stimulatesthe transcription of exocrine-specificgenes and regulates the formation ofpancreatic acinar cells (Krapp et al.,1998. Kawaguchi et al., 2002). Theloss of Rbp-j itself might directly leadto a decrease in the transcriptionalactivity of Ptf1a, because Rbp-j bindsPtf1a and further stimulates its tran-scriptional activity (Obata et al.,2001). The disturbed pancreatic exo-crine differentiation is also reminis-cent of mice lacking Hes1. Mice lack-ing Hes1 display morphologicallyimmature acinar cells with fewer se-cretory granules than are observed inthe control (Jensen et al., 2000). Hes1represses Ptf1a expression early in fo-regut development and define the nor-mal boundaries of the pancreatic do-main (Fukuda et al., 2006). However,the precise role of Notch/Rbp-j/Hes1signaling during mid-pancreatic de-velopment has been unknown. Our ob-servations indicate that, after pancre-atic commitment has been induced byPtf1a, Rbp-j supports the expressionof both Hes1 and Pdx1 and facilitatesthe differentiation of acinar cells.

The catch-up growth of pancreaticexocrine cells has been observed notonly in our pancreatic Rbp-j–deficientmice, but also in Hes1-deficient mice(Jensen et al., 2000; Fujikura et al.,2006). Stimulation of Notch signalinghas been reported to diminish the pro-

Fig. 8. No signs of endocrine or exocrine pancreatic insufficiency in Rbp-jf/f Ptf1a.cremice. A,B:Body weight (A) and morning-fed blood glucose concentrations (B) of male Rbp-jf/f Ptf1a.cremiceand control littermates at 13 weeks of age. C: Morning-fed plasma insulin concentrations of maleRbp-jf/f Ptf1a.cremice and control littermates at 15 weeks of age. D: Glucose challenge of maleRbp-jf/f Ptf1a.cremice and control littermates at 14 weeks of age. Blood glucose levels are shownat indicated times after intraperitoneal (i.p.) glucose injections (2 g/kg body weight). There were nosignificant differences (P � 0.05) between Rbp-jf/f Ptf1a.cremice and control mice at any giventime. E: Amylase activity measured in serum from male Rbp-jf/f Ptf1a.cremice and control litter-mates at 15 weeks of age. Standard errors and the number of mice examined are indicated on theaverage bars. Levels of significance (Student’s t-tests) are shown.

Fig. 6. Normal endocrine tissue in pancreas of adult Rbp-jf/f Ptf1a.cre mice. A: X-gal staining ofpancreatic regions from Rosa26Rf/� Rbp-jf/f mice (left) and Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice(right) at the adult stage. The mutant adult mice have overtly normal pancreases. B: X-gal stainingof pancreatic sections from Rosa26Rf/� Rbp-jf/f mice (left) and Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice(right) at adult. Islets are encircled by dotted lines, and ducts are indicated by arrows. No mosaicpattern of recombination was seen. C: Pancreatic deletion of Rbp-j by Ptf1a.cre. Genomic DNAfrom the tail, the pancreas, and the pool of islets of Rbp-jf/f Ptf1a.cre mice were digested with SphIand hybridized with the probe. D: The ratio of pancreatic weight to body weights of male Rbp-jf/f

Ptf1a.cre mice and control littermates at 16 weeks of age. Standard errors and number of miceexamined are indicated on the average bars. E: The � and � cell masses were determined fromanti-insulin– and anti-glucagon–stained multiple sections as described in the Experimental Pro-cedures section. Standard errors and the number of mice examined are indicated on the averagebars. F: Immunohistochemical studies with antibodies to Pdx1, insulin, glucagon, somatostatin,and pancreatic polypeptide using serial pancreatic sections from Rbp-jf/f Ptf1a.cre mice andcontrol littermates at 16 weeks of age. No significant changes of pancreatic cell composition areobserved. Scale bars � 50 �m in A,B, 200 �m in C–F.Fig. 7. Small ductal remnants in pancreas of adult Rbp-jf/f Ptf1a.cremice. A: Hematoxylin–eosinstaining, immunohistochemical studies with cytokeratin and amylase of serial pancreatic sectionsfrom Rbp-jf/f, Rbp-jf/f Pdx.cre, and Rbp-jf/f Ptf1a.cremice at 20 weeks of age. Rbp-jf/f Pdx.crepancreas exhibits cystic ductal dilations, whereas Rbp-jf/f Ptf1a.cre pancreas show only a focalarea of ductal structures at the tip. B: X-gal staining of pancreatic sections from Rosa26Rf/�

Rbp-jf/f, Rosa26Rf/� Rbp-jf/f Pdx.cre, and Rosa26Rf/� Rbp-jf/f Ptf1a.cre mice (right) at adult stage.Scale bars � 200 �m.


liferation of cultured exocrine cells ina manner independent of Hes1, butrelated to Hey1, Hey2, and p21Cip1(Rooman et al., 2006). However, themechanisms of exocrine cell prolifera-tion, especially in the growing pan-creas during the perinatal period, re-quire further investigation.

After this catch-up growth, the ar-chitecture and function of the acinartissues of the mutant mice were al-most normal. Normal adult acinarcells express little or no detectable lev-els of either Pdx1 or Notch pathwaycomponents (Wu et al., 1997; Miy-amoto et al., 2003). Forced expressionof Pdx1 in acinar cells causes amarked dysmorphogenesis and meta-plasia of the exocrine pancreas (Helleret al., 2001; Miyatsuka et al., 2006).The ectopic activation of the Notchpathway has also been observed in an-imal models of acinar cell metaplasiaand in human pancreatic cancer (Miy-amoto et al., 2003). Reduced Pdx1 ex-pression and silent Rbp-j–mediatedNotch signaling in our mutant mice iscompatible with the proposition thatthe down-regulation of Pdx1 andNotch signaling is required for theproper maintenance of the exocrinecell phenotype. Another recent studyhas shown that, in adult acinar cells,Ptf1a protein associates exclusivelywith Rbp-L, a paralog of Rbp-j, andthat the Ptf1a/Rbp-L complex stimu-lates the promoter activities of acinar-specific genes more effectively thandoes the Ptf1a/Rbp-j complex (Beres etal., 2006). The findings of our studiesindicate that Rbp-j–mediated Notchsignaling is not essential for the func-tion of mature acinar cells after theyhave fully differentiated.

Focal areas of ductal differentiationand more widespread areas of ductalstructures with prominent cysticchanges were detected in Rbp-jf/f

Ptf1a.cre and Rbp-jf/f Pdx.cre mice, re-spectively. Like Rbp-jf/f Pdx.cre mice,mice with hypomorphic Pdx1 allelesdevelop a pancreatic ductal cyst linedwith a simple cuboidal epithelium,which is positive for cytokeratin butnegative for Nkx6.1, a � cell differen-tiation marker (Fujitani et al., 2006).The inhibition of branching morpho-genesis by the depletion of Pdx1 hasbeen correlated with decreased acinarcell formation, and the residual cellsadopt the phenotype of ductal cells

(Hale et al., 2005). These results indi-cate that Rbp-j blocks the transitionfrom pancreatic progenitor epitheliumto ductal epithelium by maintainingthe expression level of Pdx1. Amongthe Notch receptors, Notch2 isstrongly expressed in the pancreaticductal cells from the embryo to theadult (Lammert et al., 2000, and ourunpublished data). We have alsogenerated pancreas-specific Notch2knockout mice. However, we havedetected no differences between thecontrol and the knockout mice (un-published data). The physiological sig-nificance of the expression of theNotch receptor in pancreatic ductalcells requires further investigation.

All our stage-specific knockout micegenerated using the Pdx1, Ptf1a, andinsulin 2 promoters suggest that Rbp-j–mediated Notch signaling is essen-tial for the expansion of early pancre-atic progenitors by maintaining theexpression of both Hes1 and Pdx1, butis not essential for mature pancreaticcell functions.

EXPERIMENTALPROCEDURES

Generation of Pancreas-Specific Rbp-j KnockoutMice

The generation of mice bearing afloxed allele of Rbp-j was describedpreviously (Han et al., 2002). Ptf1a.cremice in which the Cre recombinasegene was knocked-in into the Ptf1agene locus were reported previously(Kawaguchi et al., 2002). Mice ho-mozygous for the floxed Rbp-j allele(Rbp-jf/f) were crossed with Ptf1a.cremice. The double heterozygous micewere then crossed with Rbp-jf/� mice,resulting in Rbp-jf/f Ptf1a.cre mice andtheir control littermates. Genotypingand assessment of deletion efficiencywere performed by Southern blotanalyses on genomic DNA obtainedfrom tails, pancreases, and pool of is-lets. Other pancreas-specific Rbp-jknockout mice by using Pdx.cre trans-genic mice were recently reported(Gu et al., 2002; Fujikura et al., 2006).Rosa26-lacZ reporter mice were pur-chased from The Jackson Laboratory(Bar Harbor, ME; Soriano, 1999).

X-gal Staining

For visualization of Pdx.cre- orPtf1a.cre-mediated loxP recombina-tion, mice heterozygous for floxedRbp-j and homozygous for Rosa26Ralleles, Rosa26Rf/f Rbp-jf/�, werecrossed with double heterozygousRbp-jf/� Pdx.cre or Rbp-jf/� Ptf1a.cremice. Genotyping of floxed Rosa26Ralleles was performed by DNA dot blotanalyses on genomic DNA obtainedfrom tails or yolk sacs. A 1.2-kb ClaI–SacI LacZ fragment was used as aprobe to detect the floxed Rosa26R al-lele. Whole embryos or pancreatic re-gions were fixed with 4% paraformal-dehyde in phosphate-buffered saline(PBS) for 1 hr at 4°C and washed withPBS. To prepare cryostat sections,pancreases were dissected, fixed in 4%paraformaldehyde in PBS for 1 hr at4°C, and frozen in OCT compound(Sakura, Tokyo, Japan). Ten-micro-meter sections were cut, air-dried for30 min, and then post-fixed for 5 minin 4% paraformaldehyde. Fixed em-bryos, pancreatic regions, or tissuesections were incubated at 30°C in asolution containing PBS, potassiumferricyanide, potassium ferrocyanide,MgCl2, and galactopyranoside dis-solved in dimethyl sulfoxide. Afterstaining with X-gal, E9.5 and E10.5embryos were dehydrated in methanoland cleared in a 2:1 solution of benzylbenzoate:benzyl alcohol. Macroscopicpictures were taken using an Axios-kop Microscope (Carl Zeiss, Jena, Ger-many). X-gal–positive cells were iden-tified by their blue staining.

ImmunohistochemicalAnalyses

Embryos or isolated pancreases werefixed with 4% paraformaldehyde inPBS for overnight at 4°C, then paraf-fin embedded, and 5-�m sections werecut and mounted on glass slides.Slides were de-waxed and rehydrated.Except for the detection of hormones,the mounted sections were subjectedto antigen retrieval by autoclaving at121°C for 5 min with 10 mM citratebuffer. Endogenous peroxidase wasinactivated with 0.3% H2O2 in meth-anol for 30 min. The slides wereblocked for 1 hr at room temperaturewith a reagent containing casein(DAKO Protein Block Serum-Free So-


lution; Dako, Glostrup, Denmark),then stained overnight at 4°C with thefollowing primary antibodies (Abs) orlectin: guinea pig anti-insulin anti-body, 1:500 dilution (Dako); rabbit an-ti-glucagon antibody, 1:500 dilution(Dako); rabbit anti-somatostatin anti-body, 1:500 dilution (Dako); rabbit an-ti-pancreatic polypeptide antibody,1:500 dilution (Dako); rabbit anti–pan-cytokeratin antibody, 1:200 dilu-tion (Santa Cruz, Santa Cruz, CA);rabbit anti-amylase antibody, 1:1,000dilution (Sigma-Aldrich, St. Louis,MO); goat anti-amylase antibody, 1:50dilution (Santa Cruz); rabbit anti-car-boxypeptidase A antibody, 1:750 dilu-tion (BioTrend, Cologne, Germany);rabbit anti-Pdx1 antibody, 1:5,000 di-lution (Peshavaria et al., 1994); mouseanti-neurogenin 3 antibody, 1:100 di-lution (Developmental Studies Hy-bridoma Bank, Iowa, IA); rabbit anti-Hes1 antibody, 1:10,000 dilution (giftfrom Dr. R. Kageyama); rabbit anti-synaptophysin antibody, 1:50 dilution(Dako); rabbit anti–phospho-histoneH3 antibody, 1:50 dilution (Cell Sig-naling Technology, Beverly, MA); rab-bit anti-cleaved caspase-3 antibody,1:100 dilution (Cell Signaling Tech-nology); rabbit anti–Rbp-L antibody,1:200 dilution (gift from Dr. R. Mac-Donald); and biotinylated DBA (Vec-tor, Burlingame, CA). The slides werewashed with PBS the following dayand incubated for 1 hr at room tem-perature with the following secondaryantibodies: biotinylated goat anti-guinea pig IgG antibody, 1:200 dilu-tion; biotinylated goat anti-rabbitIgG antibody, 1:200 dilution; biotin-ylated horse anti-mouse IgG anti-body (all from Vector). The slideswere then incubated with avidin– bi-otin complex (ABC) reagent (Vec-tastain Elite ABC Kit; Vector) for 50min at room temperature followed bythe addition of diaminobenzidine tet-rahydrochloride (Dako) as a substrate–chromogen solution. After hematox-ylin counterstaining and dehydration,slides were mounted in mounting me-dium (MGK-S; Matsunami, Osaka,Japan) and pictures were taken usingan Axioskop Microscope (Carl Zeiss).

For immunofluorescence, Alexa488donkey anti-rabbit IgG antibody,1:200 dilution, and Alexa546 donkeyanti-goat IgG antibody, 1:200 dilution(both from Molecular Probes, Eugene,

OR), were used as secondary antibod-ies. Images were captured using aZeiss LSM 5 PASCAL confocal micro-scope (Carl Zeiss).

Quantitative Morphometryand Cell Counting

The relative numbers of Hes1, neuro-genin 3, synaptophysin, and phospho-histone H3–positive cells to Pdx1-pos-itive cells were calculated from theaverage of counts of 1,635 cells atE13.5 from each mouse. Four to sixmice were examined for each group.The endocrine cell mass at E15.5 wascalculated from the total area (pixelnumber) of synaptophysin-positivecells. The area of synaptophysin-posi-tive neuronal fibers was excluded.Four mice were used for each group,and 4 to 11 sections were examined foreach mouse. The acinar cell prolifera-tion rate at P0 was determined by theratio of the number of amylase/phos-pho-histone H3 double-positive cellsto the total number of amylase-posi-tive cells. For each group, four micewere used. An average of 19,000 cellswere counted per mouse. The endo-crine cell mass at 16 weeks of age wascalculated as the ratio of each hor-mone-positive cell area to the totalarea of the pancreas section using animage analysis program (Scion Image;Scion, Frederick, MD). For eachgroup, six mice were used. The mea-surement and calculation were donewith a total of 18 sections that hadbeen prepared from the six pancreases(three sections per mouse). The threesections from each mouse were farapart from one another. The wholearea of each section was investigated,and the percentage of insulin- or glu-cagon-positive cell area relative to thewhole pancreatic area was deter-mined. All values are expressed asmean � standard error. The statisti-cal significance was tested with Stu-dent’s t-test or Welch’s t-test.

Analysis of MetabolicParameters

Blood glucose values were determinedfrom whole venous blood taken frommouse tails using an automatic glu-cometer (Glutest Ace; Sanwa Kagaku,Aichi, Japan). Blood samples for insu-lin and amylase measurements were

taken from retro-orbital venous plex-uses. Plasma insulin levels were mea-sured using an enzyme-linked im-munosorbent assay kit (Morinaga,Kanagawa, Japan). Amylase activitywas measured according to the Cara-way method using a kit (Amylase-TestWako; Wako, Osaka, Japan). All val-ues are expressed as mean � standarderror. The statistical significance wastested with Student’s t-test.

Glucose Tolerance Test

After an overnight fast, the mice wereinjected intraperitoneally with glu-cose at 2.0 g/kg body weight. Bloodsamples were taken at various timepoints and blood glucose concentra-tion was measured with an automaticglucometer (Glutest Ace; Sanwa Ka-gaku). All values are expressed asmean � standard error. The statisti-cal significance was tested with Stu-dent’s t-test.

ACKNOWLEDGMENTSWe thank Prof. Douglas A. Melton forproviding Pdx.cre mice, Prof. Christo-pher V. E. Wright for Ptf1a.cre miceand Pdx1 antibody, Prof. RyoichiroKageyama for Hes1 antibody, andProf. Raymond MacDonald for Rbp-Lantibody. We also thank Prof. TasukuHonjo for his encouragement through-out this project.

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Rbp-j regulates expansion of pancreatic epithelial cells and their differentiation into exocrine...

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