Original Article - Diabetes · de la Sante´ et de la Recherche Me´dicale, INSERM E363 and U845,...

Original Article

Control of �-Cell Differentiation by the PancreaticMesenchymeMyriam Attali,

1Volodymyr Stetsyuk,

1Annie Basmaciogullari,

1Virginie Aiello,

1

Maria A. Zanta-Boussif,2

Bertrand Duvillie,1

and Raphael Scharfmann1

The importance of mesenchymal-epithelial interactions fornormal development of the pancreas was recognized in theearly 1960s, and mesenchymal signals have been shown tocontrol the proliferation of early pancreatic progenitorcells. The mechanisms by which the mesenchyme coordi-nates cell proliferation and differentiation to produce thenormal number of differentiated pancreatic cells are notfully understood. Here, we demonstrate that the mesen-chyme positively controls the final number of �-cells thatdevelop from early pancreatic progenitor cells. In vitro, thenumber of �-cells that developed from rat embryonic pan-creatic epithelia was larger in cultures with mesenchymethan without mesenchyme. The effect of mesenchyme wasnot due to an increase in �-cell proliferation but was due toincreased proliferation of early pancreatic duodenal ho-meobox-1 (PDX1)–positive progenitor cells, as confirmedby bromodeoxyuridine incorporation. Consequently, thewindow during which early PDX1� pancreatic progenitorcells differentiated into endocrine progenitor cells ex-pressing Ngn3 was extended. Fibroblast growth factor 10mimicked mesenchyme effects on proliferation of earlyPDX1� progenitor cells and induction of Ngn3 expression.Taken together, our results indicate that expansion ofearly PDX1� pancreatic progenitor cells represents a wayto increase the final number of �-cells developing fromearly embryonic pancreas. Diabetes 56:1248–1258, 2007

Epithelium-mesenchyme interactions play a cru-cial role during organogenesis. They are medi-ated at least in part by soluble factors producedby the mesenchyme and acting on the epithe-

lium (1). Evidence points to a crucial role for epithelial-mesenchymal interactions in cell proliferation anddifferentiation during pancreatic development (2). How-ever, the mechanisms by which the mesenchyme coordi-nates cell proliferation and differentiation to produce a

normal number of differentiated pancreatic cells are notfully understood.

The mature pancreas contains endocrine islets com-posed of cells producing hormones, such as insulin (�-cells) and glucagon (�-cells), and exocrine tissuecomposed of acinar cells producing enzymes (e.g., car-boxypeptidase-A) secreted into the intestine. The pan-creas originates from the dorsal and ventral regions of theforegut endoderm. Recently, important findings have shedlight on the processes controlling pancreatic endocrinecell development. Studies of genetically engineered miceidentified a hierarchy of transcription factors regulatingpancreas organogenesis and islet-cell differentiation (3–5).The endodermal region committed to a pancreatic fatefirst expresses transcription factor pancreatic duodenal

homeobox-1 (Pdx1). Pdx1 is detected in mouse embryoson embryonic day 8.5 (E8.5) (E9 in rats) in early pancre-atic progenitors. During adulthood, Pdx1 expression be-comes largely confined to �-cells, where it activatesinsulin gene transcription (6). Disruption of the Pdx1 genein mice or human leads to pancreatic agenesis (7,8). Thesedata indicate that Pdx1 is necessary for morphogenesisand differentiation of the pancreatic buds. Pdx1 is also anefficient marker of early pancreatic progenitor cells. Dif-ferentiation into endocrine and exocrine cells is the nextstep, and cell-tracing experiments have shown that bothendocrine and exocrine cells derive from Pdx1-expressingprogenitor cells (9,10). The transcription factor Neuroge-nin3 (Ngn3) is expressed in epithelial pancreatic progen-itor cells before endocrine differentiation and issubsequently downregulated during differentiation (11).Ngn3-deficient mice lack pancreatic endocrine cells (12).Lineage-tracing experiments have demonstrated thatNgn3-expressing cells are islet progenitors (10). Thus,Ngn3 is a marker of choice for detecting the onset ofpancreatic endocrine cell differentiation.

Pancreas differentiation is controlled by permissive sig-nals derived from adjacent mesodermal structures. Signalsfrom the notochord and dorsal aorta control the first stepsof pancreatic development (13,14). Signals from the mes-enchyme, which condenses around the underlying com-mitted endoderm, control the subsequent steps (2). Classicculture explant experiments highlighted the importance ofthe mesenchyme for exocrine-pancreas growth and differ-entiation (15,16). However, the role of the mesenchyme inregulating �-cell mass remains unclear. For example,results based on loss-of-function of genes encoding growthfactors expressed in the pancreatic mesenchyme, such asfibroblast growth factor 10 (FGF10), suggest that signalsfrom the mesenchyme directly and positively control thefinal number of �-cells (17). On the other hand, when

From the 1Faculty of Medicine, University Paris-Descartes, Institut Nationalde la Sante et de la Recherche Medicale, INSERM E363 and U845, NeckerHospital, Paris, France; and 2Genethon-Centre National de la RechercheScientifique Unite Mixte de Recherche 8115, Evry, France.

Address correspondence and reprint requests to Raphael Scharfmann,INSERM E363 and U845 Faculte Necker, 156 Rue de Vaugirard, 75015 Paris,France. E-mail: [email protected].

Received for publication 15 September 2006 and accepted in revised form22 January 2007.

Published ahead of print at http://diabetes.diabetesjournals.org on 23 Feb-ruary 2007. DOI: 10.2337/db06-1307.

BrdU, bromodeoxyuridine; FGF10, fibroblast growth factor 10; INSERM,Institut National de la Sante et de la Recherche Medicale; PDX1, pancreaticduodenal homeobox-1.

© 2007 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

1248 DIABETES, VOL. 56, MAY 2007

FGF10 was overexpressed in the pancreas, �-cell develop-ment was inhibited (18,19). Thus, the exact effect ofmesenchymal signals on �-cell development remains con-troversial, because studies of loss and gain of FGF10function suggest opposite conclusions.

Here, we further dissected the role of the pancreaticmesenchyme on �-cell development. This required an invitro model permissive for �-cell development both withand without mesenchyme. Previously, we had shown thatin collagen gel, �-cells developed properly from embryonic

FIG. 1. In vitro development of rat embryonic pancreatic rudiments cultured at the air/liquid interface. E13.5 rat embryonic pancreatic epitheliawere dissected with or without their mesenchyme and cultured for different periods. A–J: Representative pictures after 0 (A and F), 1 (B and G),3 (C and H), 5 (D and I), and 7 (E and J) days of culture. In F–J, the epithelium is circled. Scale bar � 50 �m. K: Representative real-time PCRanalyses of Nkx6.2, Ngn3, Pax4, NeuroD, Pcsk1, Pcsk2, and Abcc8 mRNA expression after 0, 1, 3, 5, or 7 days of culture. On each graph, geneexpression is presented as a percentage of the highest sample. Each point represents the mean � SE of three individual pools.

M. ATTALI AND ASSOCIATES

DIABETES, VOL. 56, MAY 2007 1249

pancreatic epithelium when cultured without mesen-chyme (20), whereas this culture condition was poorlypermissive for �-cell development when the epitheliumwas cultured with mesenchyme (21). Here, we developedan in vitro model in which �-cells can develop both withand without mesenchyme. Using this model, we showedthat more �-cells developed from epithelium cultured withmesenchyme than without mesenchyme. We next investi-gated whether mesenchyme increased �-cell mass byactivating �-cell proliferation or by acting on earlierevents. We found that �-cell proliferation was not modifiedby mesenchymal signals, whereas the proliferation of earlyPDX1� progenitor cells was strongly increased in thepresence of mesenchyme. Consequently, the window dur-ing which early PDX1� progenitors differentiate into en-docrine progenitors expressing Ngn3 was extended andthe amplitude of Ngn3 expression was increased. FGF10mimicked the effects of the mesenchyme on proliferationof early PDX1� progenitor cells and on induction of Ngn3expression. Thus, �-cell development is enhanced bysignals from the mesenchyme that positively control theproliferation of early embryonic pancreatic progenitor cells.

RESEARCH DESIGN AND METHODS

Pregnant Wistar rats were purchased from the Janvier breeding center (Centred’Elevage Rene Janvier, Le Genest, France). Pregnant female rats at 13.5 daysof gestation were killed by CO2 asphyxiation, according to the guidelines ofthe French Animal Care Committee. To study cell proliferation in vivo,pregnant rats at E13.5 and E18.5 were injected with bromodeoxyuridine(BrdU) (100 �g/g body wt) (Sigma) and killed 1 h later.Dissection of pancreatic rudiments. The embryos were harvested on E13.5.The stomach, the pancreas, and a small portion of the intestine were dissectedtogether; then, either the pancreatic primordium was dissected or the mesen-chyme was separated from the pancreatic epithelium as described previously (21).Organ culture. Dorsal pancreatic rudiments with or without their mesen-chyme were laid on Millicell culture plate inserts (Millipore) in 35-mm sterilePetri dishes containing 2 ml RPMI 1640 (Invitrogen) supplemented withpenicillin (100 units/ml), streptomycin (100 �g/ml), HEPES (10 mmol/l),L-glutamine (2 mmol/l), nonessential amino acids (1�; Gibco), and 10%heat-inactivated calf serum (Hyclone). Under such culture conditions, theexplants grew at the air/medium interface. Cultures were maintained at 37°Cin humidified 95% air/5% CO2. The medium was changed every other day.Recombinant human FGF10 (50 ng/ml; R&D Systems) was used in thepresence of heparin (50 �g/ml; Sigma). For cell proliferation, BrdU (10 �mol/l)was added to the culture medium.Immunohistochemistry. Tissues were fixed in 10% formalin and processedfor immunohistochemistry, as described previously (21,22). The following

FIG. 2. �-Cell development from epithelia grown with or without mesenchyme. A–J: Immunohistological analysis of pancreatic epithelia grownin vitro without (A–E) or with (F–J) mesenchyme for 0 (A and F), 1 (B and G), 3 (C and H), 5 (D and I), and 7 days (E and J). �-Cell developmentwas evaluated using anti-insulin (red) staining. Nuclei were stained in blue with Hoechst. K: Quantification of the absolute surface areas occupiedby insulin� cells that developed with or without mesenchyme. Three rudiments were analyzed for each condition. Data are means � SE. **P <0.01. Scale bar � 50 �m.

MESENCHYME AND �-CELL DIFFERENTIATION


antibodies were used: mouse anti-human insulin (1/2,000; Sigma), rabbitanti-insulin (1/2,000; Diasorin), mouse anti-BrdU (1/2; Amersham), and rabbitanti-PDX1 (1/1,000) (22). The fluorescent secondary antibodies were fluores-cein isothiocyanate anti-rabbit and Texas-red anti-mouse antibodies (1/200;Jackson Immunoresearch) and Alexa-fluor anti-rabbit antibody (1/400; Bio-genex). Photographs were taken using a fluorescence microscope (LeitzDMRB; Leica) and digitized using a C5810 cooled 3CCD camera (Hamamatsu,

Middlesex, NJ). No signals were observed when the first antibodies wereomitted.In situ hybridization. Tissues were fixed at 4°C in 4% paraformaldehyde inPBS and either included in paraffin or cryoprotected in 15% sucrose-PBS at4°C overnight, embedded in 15% sucrose-7.5% gelatin in PBS, and frozen inisopentane. Paraffin sections (4 �m thick) or cryosections (14 �m thick) wereprepared. A Ngn3 probe (726 bp) was used (23) and in situ hybridization was

FIG. 3. The mesenchyme does not activate �-cell proliferation. A–F: Pancreatic epithelia were grown for 3 (A and D), 5 (B and E), or 7 (C andF) days without (A–C) or with (D–F) mesenchyme and pulsed with BrdU during the last hour of culture. Insulin staining is in red and BrdU ingreen. G: Quantification of the number of �-cells in S phase on days 3, 5, and 7, indicating that the mesenchyme did not activate �-cellproliferation. H: For �-cell proliferation in vivo, pregnant rats were injected with BrdU on E18.5 and killed 1 h later. Pancreases wereimmunostained for insulin (red) and BrdU (green). Scale bar � 50 �m.



done as previously described (24). No signal was obtained when a senseriboprobe was used.Quantification. To quantify the absolute numbers of insulin- and Ngn3-expressing cells, all sections of each pancreatic rudiment were digitized. Onevery image, the surfaces of insulin or Ngn3 stainings were quantified usingIPLab Eval (version 3.2.4; Scanalytics), and the stained areas were summed aspreviously described (25). Three to four rudiments were analyzed per condition.

To measure proliferation of either �-cells or early progenitors expressingPDX1, we counted the frequency of BrdU� nuclei among 1,000 insulin� cellsor 1,000 early PDX1� progenitors per rudiment. Three rudiments wereanalyzed per condition.

To quantify the maintenance of early PDX1� progenitors, we counted thenumber of insulin� cells among 1,000 PDX1� cells. The percentage of undiffer-

entiated PDX1-expressing cells was then calculated. Three rudiments wereanalyzed per condition. Statistical significance was determined using Student’s t

test.RNA extraction and real-time PCR. Total RNA was extracted from pools ofthree to four embryonic pancreases (cultured with or without mesenchyme)using Rneasy microkit (Qiagen). The cDNAs were generated using Superscriptreagents (Invitrogen) according to the manufacturer’s instructions. Real-timePCR was performed using a 7300 real-time PCR system (Applied Biosystems).Each reaction consist of a mix of Taqman universal PCR master mix (AppliedBiosystems) with primers and Taqman-labeled probe specific for each gene(Applied Biosystems) and run after universal thermal cycling protocol (95°Cfor 10 min followed by 40 cycles of 95°C for 15 s and 65°C for 1 min). Controlreactions in the absence of template were included in each assay. Results

FIG. 4. The mesenchyme amplifies the early PDX1� cell pool. A–B: Pancreatic epithelia were grown for 24 h without (A) or with (B and B�)mesenchyme and pulsed with BrdU during the last hour of culture. Immunohistochemistry was performed using anti-PDX1 (in green) andanti-BrdU (in red) antibodies. Nuclei were stained in blue with Hoechst. B� is an enlargement of B. C: Quantification of the number of early PDX1�

cells in S phase after 24 h of culture. Data are means � SE. **P < 0.01. D and D�: In vivo proliferation of early PDX1� progenitors. Pregnant ratswere injected with BrdU on E13.5 and killed 1 h later. Immunostaining for PDX1 is in green and BrdU in red. D� is an enlargement of D. Scale bar �50 �m.



were normalized to the transcript encoding the housekeeping protein cyclo-philin A. Each point represents the mean � SE of three individual pools. PCRprimer sequences are available on request.

RESULTS

Development of rat embryonic pancreatic epitheliumcultured at the air/liquid interface with or without itsmesenchyme. We developed a culture model that allowed�-cell differentiation with and without mesenchyme. Forthis purpose, we dissected pancreatic epithelia at E13.5. Atthis stage, the pancreatic epithelium is mainly composedof early PDX1� progenitors, with few glucagon� endo-crine cells. We cultured pancreatic rudiments at the air/liquid interface on filters floating on culture medium.Without mesenchyme, the epithelium acquired a sphericalshape after 1 day in culture, and the size of the tissues didnot change significantly during the 7-day culture period(Fig. 1A–E). At the end of the 7 days, the epitheliumappeared as a small dark sphere surrounded by a variablenumber of translucent buds (Fig. 1E). In the presence ofmesenchyme, in contrast, the epithelium grew rapidly,spread into the mesenchyme, and developed lobules (Fig.1F–J).

We next analyzed the in vitro pattern of expression ofdifferent pancreatic genes. We focused on Nkx6.2 ex-

pressed in vivo at early stages of development, its expres-sion declining in late embryogenesis (26), Ngn3 and Pax4

known to be expressed in pancreatic endocrine progeni-tors, and NeuroD expressed both in endocrine progenitorsand in mature �-cells. We also followed the expression ofthree genes expressed in mature �-cells: Pcsk1, Pcsk2, andAbcc8. As shown in Fig. 1K, Nkx6.2 was expressed duringthe first 3 days of culture, its expression decreasingthereafter. The expression of Ngn3 and Pax4 was turnedon and next decreased both with and without mesen-chyme, as expected for markers of pancreatic progenitors.The expression of NeuroD was rapidly activated and didnot decrease with time, as expected for a gene expressedboth in progenitors and mature �-cells. Finally, the expres-sion of Pcsk1, Pcsk2, and Abcc8 increased at late timepoints of in vitro development.Effect of the mesenchyme on the absolute number of

�-cells. We first compared �-cell development from epi-thelia cultured with or without mesenchyme. In epitheliacultured with or without mesenchyme, the first insulin-expressing cells were detected after 3 days of culture (Fig.2C and H), and their number increased thereafter (Fig. 2D,E, I, and J). We next compared �-cell development in bothconditions. As shown in Fig. 2K, both with and without

FIG. 5. The pool of early PDX1� progenitor cells is maintained in the presence of mesenchyme. A–I: Immunohistological analyses of E13.5 ratpancreatic epithelia cultured for 0 (A), 1 (B and F), 3 (C and G), 5 (D and H), or 7 (E and I) days without (B–E) or with (F–I) mesenchyme.Immunostaining for PDX1 is in green and insulin in red. J: Quantification of the proportion of PDX1� cells that remained negative for insulin.Three rudiments were analyzed for each condition. Data are means � SE. *P < 0.05. Scale bar � 50 �m.



mesenchyme, the number of �-cells increased during thefirst 5 culture days. However, the number of �-cells did notincrease further from days 5 to 7 without mesenchyme butcontinued to increase with mesenchyme. As a result, asignificantly larger number of �-cells developed with thanwithout mesenchyme. Individual �-cell size did not vary inthe different culture conditions (data not shown). Whenepithelium was dissected, directly reassociated with mes-enchyme, and cultured, its development was similar to theone of whole pancreas (data not shown).The effect of the mesenchyme on the number of�-cells that develop is not due to an increase in �-cellproliferation. We compared BrdU incorporation in�-cells developed without or with the mesenchyme. After3 days of culture, �-cells developed without or withmesenchyme did not incorporate BrdU (Fig. 3A and D).After 5 and 7 days of culture, some �-cells developedwithout mesenchyme incorporated BrdU (Fig. 3B and C);this proliferation was even lower with mesenchyme (Fig.3E–G for quantification). This low proliferation of �-cellsresembles the one found in the rat embryonic pancreas atE18.5 (Fig. 3H). Thus, the increase in the number of �-cellsthat developed with mesenchyme cannot be explained byan activation of �-cell proliferation by the mesenchyme.

The mesenchyme activates the proliferation of early

pancreatic progenitors. We cultured epithelia for 1 daywith or without mesenchyme and added BrdU during thelast hour of culture. The percentage of PDX1� cells thatincorporated BrdU was 5 � 2% without mesenchyme (Fig.4A) and 39 � 7% with mesenchyme (Fig. 4B, B�, and C forquantification). When epithelium was dissected, directlyreassociated with mesenchyme, and cultured for 1 day, theproliferation of early PDX1� progenitors was similar to theone of whole pancreas (data not shown). A similar highlevel of PDX1�-cell proliferation in the presence of mes-enchyme can be found in the E13.5 rat embryonic pan-creas (Fig. 4D and D�). Thus, the mesenchyme is necessaryfor maintaining the proliferation of early pancreatic pro-genitors.The pool of early PDX1� progenitors is maintained inthe presence of mesenchyme. To investigate the conse-quences of proliferation of early PDX1� pancreatic pro-genitors, we quantified the maintenance of these cells withor without mesenchyme. We cultured pancreatic epitheliawith or without mesenchyme and quantified the decreasein the number of early PDX1�/insulin� progenitors. AtE13.5, the epithelium is composed of early PDX1�/insu-lin� cells (Fig. 5A). In cultures without mesenchyme (Fig.

FIG. 6. Expression of Ngn3 in embryonic pancreatic epithelia cultured with or without mesenchyme. A–K: E13.5 rat pancreatic epithelia culturedfor 0 (A), 1 (B and G), 3 (C and H), 5 (D and I), 7 (E and J), or 9 (F and K) days without (B–F) or with (G–K) mesenchyme. Ngn3 expressionwas detected by in situ hybridization. L: Quantification of the absolute surface areas occupied by Ngn3� cells that developed over 0, 1, 3, 5, 7,or 9 days of culture with or without mesenchyme. Three to four rudiments were analyzed for each condition. Data are means � SE. **P < 0.01.Scale bar � 50 �m.



5B–E and J for quantification), the proportion of earlyPDX1�/insulin� progenitors decreased rapidly, and after 3and 5 days, 41 � 4 and 23 � 5% of PDX1� cells remainedinsulin negative, respectively. With mesenchyme (Fig.5F–I and J for quantification), the decrease in the propor-tion of early PDX1�/insulin� progenitors was slower, andafter 3 and 5 days, 76 � 4 and 51 � 11% of PDX1� cellsremained insulin negative, respectively. By knowing theabsolute area occupied by �-cells and the percentage ofundifferentiated Pdx1� cells, we deduced and comparedthe absolute number of undifferentiated Pdx1� cells. Wefound 4.02 and 4.37 times more PDX1� insulin� cells withthan without mesenchyme after 3 and 5 days of culture,respectively. These results indicate that, in addition toincreasing progenitor cell proliferation, the mesenchymemaintains a pool of early PDX1� progenitors.The increase in the number of early PDX1� progeni-tors by the mesenchyme enhances the �-cell pathway.We next monitored Ngn3 expression in epithelia grownwith or without mesenchyme. At E13.5, very few cellsexpressed Ngn3 in rat pancreatic epithelium (Fig. 6A).After 1 day of culture without mesenchyme, Ngn3 expres-sion increased dramatically, reaching a peak followed by aplateau until day 3 then by a sharp decrease on days 5 and7 (Fig. 6B–F). With mesenchyme (Fig. 6G–K), the numberof Ngn3� cells increased slightly at day 1 compared withepithelia cultured alone, but interestingly, in 3- and 5-day-old cultures, the number of Ngn3� cells was dramaticallyhigher in epithelia cultured with than without mesen-chyme (Fig. 6L). In addition, Ngn3 expression was pro-longed with mesenchyme: Whereas Ngn3� cells wereextremely scarce after 5 days and undetectable after 7days without mesenchyme, they were present after 5 and7 days of culture with mesenchyme. Compare Fig. 6D with

I, and compare Fig. 6E with J. Quantifications are shownin Fig. 6L.

Hypotheses to explain the increase in the number ofNgn3� cells in the presence of mesenchyme includeincreased differentiation of amplified PDX1� progenitorsand proliferation of Ngn3� cells. To test the latter hypoth-esis, we measured BrdU incorporation by Ngn3� cellsgrown in vitro from epithelia cultured for 3 or 5 days withor without mesenchyme. Extremely rare Ngn3� cellsincorporating BrdU could be found in epithelia that devel-oped either with or without mesenchyme (Fig. 7). Thispoor in vitro proliferation of Ngn3� cells is reminiscent ofthe low level of Ngn3�-cell proliferation in the pancreas atE16.5 and E18.5 (data not shown) and could not explainthe major increase in the number of Ngn3� cells in thepresence of mesenchyme. Thus, our data strongly suggestthat the mesenchyme may amplify Ngn3 expression byincreasing the magnitude and duration of development ofearly PDX1� progenitors into Ngn3� progenitors.FGF10 mimics the effects of the mesenchyme onproliferation and differentiation of early PDX1� pro-genitors. In mice, Fgf10 is expressed in the mesenchymeadjacent to the early pancreatic epithelial buds and re-quired for normal development of the pancreas (17). In rat,FGF10 is expressed in the pancreatic mesenchyme be-tween E12.5 and E18 (27; data not shown). We thus testedwhether the effects of the mesenchyme on early progenitorcell proliferation and differentiation could be mimicked byFGF10. Epithelia were cultured for 1 day with or withoutFGF10. As shown in Fig. 8A–C, FGF10 treatment signifi-cantly increased the proliferation of PDX1� progenitors.To test the effect of FGF10 on progenitor cell differentia-tion, epithelia were cultured for 1, 3, or 5 days alone, withmesenchyme, or with FGF10. Without mesenchyme andFGF10, Ngn3 expression increased after 1 day of culture,reached a plateau at day 3, and decreased thereafter. Onthe other hand, in the presence of either mesenchyme orFGF10, the pattern of expression of Ngn3 was amplifiedand prolonged (Fig. 8D–M). For example, whereas after 5days of culture, extremely rare Ngn3� cells were detectedin epithelia cultured alone (Fig. 8G), Ngn3� cells wereabundant in epithelia cultured with FGF10 (Fig. 8M). Sucheffects of FGF10 on Ngn3 expression were further con-firmed by real-time PCR (data not shown).

Finally, we quantified the absolute number of �-cellsdeveloped in epithelia cultured for 5 days in the absence orpresence of FGF10. As shown in Fig. 8N–P, FGF10 treat-ment significantly increased the absolute number of�-cells that develop. Such effect of FGF10 was also con-firmed by real-time PCR (data not shown).

DISCUSSION

Our results show that the mesenchyme positively regu-lates the final number of �-cells developed from embry-onic pancreas. This effect was not due to a direct action ofthe mesenchyme on the proliferation of either �-cells orNgn3� endocrine progenitors. Instead, the mesenchymeacted by increasing the proliferation of early PDX1�

pancreatic progenitors and amplifying and prolonging theformation of Ngn3� endocrine progenitors.

We used an in vitro model that allows �-cell develop-ment from embryonic pancreatic epithelium cultured withand without mesenchyme (28). In earlier work, we showedthat �-cells developed from embryonic pancreatic epithe-lium cultured in collagen gel without mesenchyme (20),

FIG. 7. The mesenchyme does not activate the proliferation of Ngn3-expressing progenitor cells. Pancreatic epithelia were grown without(A and B) or with (C and D) mesenchyme for 3 (A and C) or 5 (B andD) days and pulsed with BrdU during the last hour of culture. Ngn3

expression was detected by in situ hybridization (in blue), whereasBrdU� cells were shown in green. Scale bar � 25 �m.



whereas �-cells developed poorly when cultured withmesenchyme (21). This inhibitory effect of mesenchymewas due to delayed Ngn3 induction and required a func-tional Notch pathway (24). A working hypothesis was thathypoxia occurred in collagen gel, activating the Notchpathway and inhibiting endocrine differentiation, a mech-anism recently established for other cell types (29).

Here, we cultured rat embryonic pancreatic epithelia atthe air/liquid interface. Under these conditions, �-cellsdeveloped in epithelia cultured with or without mesen-chyme. �-Cells never developed from mesenchyme cul-

tured alone (data not shown). We then found that �-celldevelopment in this in vitro model mimicked in vivodevelopment. For example, Ngn3 expression was turnedon rapidly then turned off a few days later, in keeping withthe reported in vivo pattern (11,30). Also similar to in vivoevents, in vitro, Ngn3 induction was followed by theactivation of other genes, such as Pax4 and NeuroD, andfinally by the induction of markers of terminally differen-tiated �-cells, such as Pcsk1 and Pcsk2, two endoproteo-lytic enzymes necessary for proinsulin processing, andAbcc8, a sulfonylurea receptor (31–33). It is also interest-

FIG. 8. FGF10 mimics the effects of the mesenchyme on proliferation and differentiation of early PDX1� progenitor cells. A--C: Pancreaticepithelia were grown without (A) or with (B) FGF10 for 1 day and pulsed with BrdU during the last hour of culture. Immunostaining for PDX1is in green and BrdU in red. C: Quantification of the number of PDX1� cells in S phase. Data are means � SE. **P < 0.01. Scale bar � 50 �m. D–M:E13.5 rat pancreatic epithelia cultured for 0 (D), 1 (E, H, and K), 3 (F, I, and L), or 5 (G, J, and M) days without (E–G) or with (H–J) mesenchymeor without mesenchyme but with FGF10 (K–M). Ngn3 expression was detected by in situ hybridization (in blue). Scale bar � 50 �m. N–P: In vitroeffects of FGF10 on �-cell development. Pancreatic epithelia were grown without (N) or with (O) FGF10 for 5 days. Insulin� cells were revealedin red. Nuclei are stained in blue with Hoechst. P: Quantification of the absolute surface areas occupied by insulin� cells that developed during5 days without or with FGF10. Three rudiments were analyzed for each condition. Data are means � SE. **P < 0.01. Scale bar � 50 �m.



ing to note that in vitro cell proliferation faithfullyreplicated in vivo events (17,26,34). Specifically, withmesenchyme, early PDX1� progenitors proliferated at afast pace. When the cells entered the endocrine pathwayand expressed Ngn3, proliferation decreased sharply. Fi-nally, the first �-cells that developed after 3 days of culturedid not proliferate. This simple in vitro model in whichproliferation and differentiation mimic in vivo events isextremely useful for elucidating the effects of the mesen-chyme at each step of �-cell development.

The exact role for the mesenchyme in coordinatingprogenitor cell proliferation and differentiation is incom-pletely understood (15,16). Here, we demonstrate that themesenchyme increases the proliferation of early PDX1�

pancreatic progenitors, amplifying and prolonging the for-mation of Ngn3� endocrine progenitors and ultimately in-creasing �-cell development, and FGF10 mimics the effectof the mesenchyme. However, FGF10 does not providecomplete growth of the explants when compared withmesenchyme. We are currently testing whether in additionto FGF10, the mesenchyme generates additional signalsthat are important for pancreatic epithelial development.

We previously demonstrated that FGF10 is produced byembryonic pancreatic mesenchymal cells and is requiredfor the proliferation of early pancreatic progenitors (17).We suggested that failure of the progenitor pool to expandin Fgf10 mutants might lead to a reduced number ofprogenitors available for differentiation, ultimately result-ing in an inadequate number of cells to produce a normalpancreas. However, the detrimental effects of FGF10 de-letion hindered the interpretation of the role for FGF10 inlater stages of development (17). However, in transgenicmice ectopically expressing FGF10 in the pancreatic epi-thelium, epithelial cell proliferation increased but pancre-atic differentiation of all cell types was markedlydecreased (18,19). Such a phenotype was also observedwhen E10 mouse pancreatic epithelium was cultured inmatrigel in the presence of FGF10 (35). This inhibition ofdifferentiation was unexpected, and the contradiction be-tween the phenotype of FGF10-deficient mice and trans-genic mice with ectopic FGF10 expression or E10 mousepancreatic epithelium grown with FGF10 was unex-plained. For example, the phenotype of FGF10-deficientmice suggested that FGF10 did not inhibit endocrine celldifferentiation. In the absence of FGF10, there was noexcessive expression of early markers for endocrine cells,such as Isl1 and Ngn3. Moreover, glucagon-expressingcells did not form prematurely or in excess (17). Incontrast, endocrine cell differentiation was strongly inhib-ited in transgenic mice overexpressing FGF10 or in E10mouse pancreatic epithelium grown with FGF10(18,19,35). One hypothesis for explaining these apparentcontradictions involves the timing of FGF10 expression intransgenic mice. In these mice, FGF10 expression iscontrolled by the Pdx1 promoter, which is expressed veryearly in the pancreas, before the mesenchyme condensesaround the gut tube. At this early stage, FGF10 treatmentof dorsal pancreatic endoderm explants was sufficient toincrease the expression of Ptf1a, a transcription factorneeded to complete pancreatic specification (36,37). Thus,one possibility is that early FGF10 misexpression in thepancreas modifies the pancreatic cell-differentiation pro-gram, a mechanism recently suggested to explain thepancreatic phenotype of mice that overexpress Wnt1under the control of the Pdx1 promoter (38). These micehave pancreatic agenesis. Comparisons of their phenotype

with that of mice deficient in �-catenin suggest that Wnt1overexpression at early development stages may reflectalternative roles for Wnt signaling, such as specifying thefate of the intestine (39). Difference in the stage ofdevelopment of the explants could also explain the differ-ences between our study and the one by Miralles et al.(35). We used E13.5 rat pancreas, whereas Miralles et al.used E10 mouse pancreas. Culture conditions could alsoexplain the differences. Although, here, we grew explantson filters at the air/liquid interface, Miralles et al. per-formed cultures in matrigel that contains basement mem-brane proteins that are not fully characterized.

Taken together, our data indicate that 1) the pancre-atic mesenchyme increases the final �-cell population byactivating the proliferation of early PDX1� pancreaticprogenitors; and 2) the effects of the mesenchyme onproliferation and differentiation can be mimicked byFGF10. We propose that early PDX1� pancreatic progen-itors represent an expansion pool characterized by asignificant proliferative potential that should be exploitedfor expansion with FGF10 and differentiation into �-cells.Interestingly, Baetge and colleagues (40) recently directeddifferentiation of human embryonic stem cells into insulin-producing cells by mimicking embryonic development.However, as noted by the authors, the protocol must befurther improved to produce therapeutic �-cells. The in-formation we provide here should be useful to define theexact timing during which FGF10 should be used toamplify pancreatic progenitors and thus increase the finalnumber of �-cells that will develop.

ACKNOWLEDGMENTS

M.A. received support from the French Ministry for Re-search and Technology and from the Association Pour laRecherche Medical. This work was supported by JuvenileDiabetes Research Foundation (JDRF Center for CellTherapy in Europe), INSERM/Fondation pour la Recher-che Medicale/JDRF (Grant 4DA03H), the National Insti-tutes of Health Beta Cell Biology Consortium (DK 072495-02), the 6th European Union Framework Program (�-CellTherapy Integrated Project), INSERM-JDRF Grant (AIPCellules souches A03139MS), the French National Pro-gram of Research on Diabetes, and the Association Fran-caise des Diabetiques.

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