Epithelium-mesenchyme interactions play a crucial. Original Article Control of -Cell Differentiation by the Pancreatic Mesenchyme

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1 Original Article Control of -Cell Differentiation by the Pancreatic Mesenchyme Myriam Attali, 1 Volodymyr Stetsyuk, 1 Annie Basmaciogullari, 1 Virginie Aiello, 1 Maria A. Zanta-Boussif, 2 Bertrand Duvillie, 1 and Raphael Scharfmann 1 The importance of mesenchymal-epithelial interactions for normal development of the pancreas was recognized in the early 1960s, and mesenchymal signals have been shown to control the proliferation of early pancreatic progenitor cells. The mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce the normal number of differentiated pancreatic cells are not fully understood. Here, we demonstrate that the mesenchyme positively controls the final number of -cells that develop from early pancreatic progenitor cells. In vitro, the number of -cells that developed from rat embryonic pancreatic epithelia was larger in cultures with mesenchyme than without mesenchyme. The effect of mesenchyme was not due to an increase in -cell proliferation but was due to increased proliferation of early pancreatic duodenal homeobox-1 (PDX1) positive progenitor cells, as confirmed by bromodeoxyuridine incorporation. Consequently, the window during which early PDX1 pancreatic progenitor cells differentiated into endocrine progenitor cells expressing Ngn3 was extended. Fibroblast growth factor 10 mimicked mesenchyme effects on proliferation of early PDX1 progenitor cells and induction of Ngn3 expression. Taken together, our results indicate that expansion of early PDX1 pancreatic progenitor cells represents a way to increase the final number of -cells developing from early embryonic pancreas. Diabetes 56: , 2007 Epithelium-mesenchyme interactions play a crucial role during organogenesis. They are mediated at least in part by soluble factors produced by the mesenchyme and acting on the epithelium (1). Evidence points to a crucial role for epithelialmesenchymal interactions in cell proliferation and differentiation during pancreatic development (2). However, the mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce a From the 1 Faculty of Medicine, University Paris-Descartes, Institut National de la Santé et de la Recherche Médicale, INSERM E363 and U845, Necker Hospital, Paris, France; and 2 Genethon-Centre National de la Recherche Scientifique Unité Mixte de Recherche 8115, Evry, France. Address correspondence and reprint requests to Raphael Scharfmann, INSERM E363 and U845 Faculté Necker, 156 Rue de Vaugirard, Paris, France. scharfmann@necker.fr. Received for publication 15 September 2006 and accepted in revised form 22 January Published ahead of print at on 23 February DOI: /db BrdU, bromodeoxyuridine; FGF10, fibroblast growth factor 10; INSERM, Institut National de la Santé et de la Recherche Médicale; PDX1, pancreatic duodenal homeobox 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. normal number of differentiated pancreatic cells are not fully understood. The mature pancreas contains endocrine islets composed of cells producing hormones, such as insulin ( cells) and glucagon ( -cells), and exocrine tissue composed of acinar cells producing enzymes (e.g., carboxypeptidase-a) secreted into the intestine. The pancreas originates from the dorsal and ventral regions of the foregut endoderm. Recently, important findings have shed light on the processes controlling pancreatic endocrine cell development. Studies of genetically engineered mice identified a hierarchy of transcription factors regulating pancreas organogenesis and islet-cell differentiation (3 5). The endodermal region committed to a pancreatic fate first expresses transcription factor pancreatic duodenal homeobox-1 (Pdx1). Pdx1 is detected in mouse embryos on embryonic day 8.5 (E8.5) (E9 in rats) in early pancreatic progenitors. During adulthood, Pdx1 expression becomes largely confined to -cells, where it activates insulin gene transcription (6). Disruption of the Pdx1 gene in mice or human leads to pancreatic agenesis (7,8). These data indicate that Pdx1 is necessary for morphogenesis and differentiation of the pancreatic buds. Pdx1 is also an efficient marker of early pancreatic progenitor cells. Differentiation into endocrine and exocrine cells is the next step, and cell-tracing experiments have shown that both endocrine and exocrine cells derive from Pdx1-expressing progenitor cells (9,10). The transcription factor Neurogenin3 (Ngn3) is expressed in epithelial pancreatic progenitor cells before endocrine differentiation and is subsequently downregulated during differentiation (11). Ngn3-deficient mice lack pancreatic endocrine cells (12). Lineage-tracing experiments have demonstrated that Ngn3-expressing cells are islet progenitors (10). Thus, Ngn3 is a marker of choice for detecting the onset of pancreatic endocrine cell differentiation. Pancreas differentiation is controlled by permissive signals derived from adjacent mesodermal structures. Signals from the notochord and dorsal aorta control the first steps of pancreatic development (13,14). Signals from the mesenchyme, which condenses around the underlying committed endoderm, control the subsequent steps (2). Classic culture explant experiments highlighted the importance of the mesenchyme for exocrine-pancreas growth and differentiation (15,16). However, the role of the mesenchyme in regulating -cell mass remains unclear. For example, results based on loss-of-function of genes encoding growth factors expressed in the pancreatic mesenchyme, such as fibroblast growth factor 10 (FGF10), suggest that signals from the mesenchyme directly and positively control the final number of -cells (17). On the other hand, when 1248 DIABETES, VOL. 56, MAY 2007

2 M. ATTALI AND ASSOCIATES FIG. 1. In vitro development of rat embryonic pancreatic rudiments cultured at the air/liquid interface. E13.5 rat embryonic pancreatic epithelia were 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 PCR analyses 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, gene expression is presented as a percentage of the highest sample. Each point represents the mean SE of three individual pools. FGF10 was overexpressed in the pancreas, -cell development was inhibited (18,19). Thus, the exact effect of mesenchymal signals on -cell development remains controversial, because studies of loss and gain of FGF10 function suggest opposite conclusions. Here, we further dissected the role of the pancreatic mesenchyme on -cell development. This required an in vitro model permissive for -cell development both with and without mesenchyme. Previously, we had shown that in collagen gel, -cells developed properly from embryonic DIABETES, VOL. 56, MAY

3 MESENCHYME AND -CELL DIFFERENTIATION FIG. 2. -Cell development from epithelia grown with or without mesenchyme. A J: Immunohistological analysis of pancreatic epithelia grown in 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 development was evaluated using anti-insulin (red) staining. Nuclei were stained in blue with Hoechst. K: Quantification of the absolute surface areas occupied by insulin cells that developed with or without mesenchyme. Three rudiments were analyzed for each condition. Data are means SE. **P < Scale bar 50 m. pancreatic epithelium when cultured without mesenchyme (20), whereas this culture condition was poorly permissive for -cell development when the epithelium was cultured with mesenchyme (21). Here, we developed an in vitro model in which -cells can develop both with and without mesenchyme. Using this model, we showed that more -cells developed from epithelium cultured with mesenchyme than without mesenchyme. We next investigated whether mesenchyme increased -cell mass by activating -cell proliferation or by acting on earlier events. We found that -cell proliferation was not modified by mesenchymal signals, whereas the proliferation of early PDX1 progenitor cells was strongly increased in the presence of mesenchyme. Consequently, the window during which early PDX1 progenitors differentiate into endocrine progenitors expressing Ngn3 was extended and the amplitude of Ngn3 expression was increased. FGF10 mimicked the effects of the mesenchyme on proliferation of early PDX1 progenitor cells and on induction of Ngn3 expression. Thus, -cell development is enhanced by signals from the mesenchyme that positively control the proliferation of early embryonic pancreatic progenitor cells. RESEARCH DESIGN AND METHODS Pregnant Wistar rats were purchased from the Janvier breeding center (Centre d Elevage René Janvier, Le Genest, France). Pregnant female rats at 13.5 days of gestation were killed by CO 2 asphyxiation, according to the guidelines of the 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 dissected together; then, either the pancreatic primordium was dissected or the mesenchyme was separated from the pancreatic epithelium as described previously (21). Organ culture. Dorsal pancreatic rudiments with or without their mesenchyme were laid on Millicell culture plate inserts (Millipore) in 35-mm sterile Petri dishes containing 2 ml RPMI 1640 (Invitrogen) supplemented with penicillin (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, the explants grew at the air/medium interface. Cultures were maintained at 37 C in humidified 95% air/5% CO 2. The medium was changed every other day. Recombinant human FGF10 (50 ng/ml; R&D Systems) was used in the presence 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 processed for immunohistochemistry, as described previously (21,22). The following 1250 DIABETES, VOL. 56, MAY 2007

4 M. ATTALI AND ASSOCIATES 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 and F) 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 in green. G: Quantification of the number of -cells in S phase on days 3, 5, and 7, indicating that the mesenchyme did not activate -cell proliferation. H: For -cell proliferation in vivo, pregnant rats were injected with BrdU on E18.5 and killed 1 h later. Pancreases were immunostained for insulin (red) and BrdU (green). Scale bar 50 m. antibodies were used: mouse anti-human insulin (1/2,000; Sigma), rabbit anti-insulin (1/2,000; Diasorin), mouse anti-brdu (1/2; Amersham), and rabbit anti-pdx1 (1/1,000) (22). The fluorescent secondary antibodies were fluorescein isothiocyanate anti-rabbit and Texas-red anti-mouse antibodies (1/200; Jackson Immunoresearch) and Alexa-fluor anti-rabbit antibody (1/400; Biogenex). Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica) and digitized using a C5810 cooled 3CCD camera (Hamamatsu, Middlesex, NJ). No signals were observed when the first antibodies were omitted. In situ hybridization. Tissues were fixed at 4 C in 4% paraformaldehyde in PBS and either included in paraffin or cryoprotected in 15% sucrose-pbs at 4 C overnight, embedded in 15% sucrose-7.5% gelatin in PBS, and frozen in isopentane. Paraffin sections (4 m thick) or cryosections (14 m thick) were prepared. A Ngn3 probe (726 bp) was used (23) and in situ hybridization was DIABETES, VOL. 56, MAY

5 MESENCHYME AND -CELL DIFFERENTIATION 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) and anti-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 < D and D : In vivo proliferation of early PDX1 progenitors. Pregnant rats were 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. done as previously described (24). No signal was obtained when a sense riboprobe was used. Quantification. To quantify the absolute numbers of insulin- and Ngn3- expressing cells, all sections of each pancreatic rudiment were digitized. On every image, the surfaces of insulin or Ngn3 stainings were quantified using IPLab Eval (version 3.2.4; Scanalytics), and the stained areas were summed as previously described (25). Three to four rudiments were analyzed per condition. To measure proliferation of either -cells or early progenitors expressing PDX1, we counted the frequency of BrdU nuclei among 1,000 insulin cells or 1,000 early PDX1 progenitors per rudiment. Three rudiments were analyzed per condition. To quantify the maintenance of early PDX1 progenitors, we counted the number of insulin cells among 1,000 PDX1 cells. The percentage of undifferentiated PDX1-expressing cells was then calculated. Three rudiments were analyzed per condition. Statistical significance was determined using Student s t test. RNA extraction and real-time PCR. Total RNA was extracted from pools of three to four embryonic pancreases (cultured with or without mesenchyme) using Rneasy microkit (Qiagen). The cdnas were generated using Superscript reagents (Invitrogen) according to the manufacturer s instructions. Real-time PCR was performed using a 7300 real-time PCR system (Applied Biosystems). Each reaction consist of a mix of Taqman universal PCR master mix (Applied Biosystems) with primers and Taqman-labeled probe specific for each gene (Applied Biosystems) and run after universal thermal cycling protocol (95 C for 10 min followed by 40 cycles of 95 C for 15 s and 65 C for 1 min). Control reactions in the absence of template were included in each assay. Results 1252 DIABETES, VOL. 56, MAY 2007

6 M. ATTALI AND ASSOCIATES FIG. 5. The pool of early PDX1 progenitor cells is maintained in the presence of mesenchyme. A I: Immunohistological analyses of E13.5 rat pancreatic 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 < Scale bar 50 m. were normalized to the transcript encoding the housekeeping protein cyclophilin A. Each point represents the mean SE of three individual pools. PCR primer sequences are available on request. RESULTS Development of rat embryonic pancreatic epithelium cultured at the air/liquid interface with or without its mesenchyme. We developed a culture model that allowed -cell differentiation with and without mesenchyme. For this purpose, we dissected pancreatic epithelia at E13.5. At this stage, the pancreatic epithelium is mainly composed of early PDX1 progenitors, with few glucagon endocrine cells. We cultured pancreatic rudiments at the air/ liquid interface on filters floating on culture medium. Without mesenchyme, the epithelium acquired a spherical shape after 1 day in culture, and the size of the tissues did not change significantly during the 7-day culture period (Fig. 1A E). At the end of the 7 days, the epithelium appeared as a small dark sphere surrounded by a variable number of translucent buds (Fig. 1E). In the presence of mesenchyme, 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 of different pancreatic genes. We focused on Nkx6.2 expressed in vivo at early stages of development, its expression declining in late embryogenesis (26), Ngn3 and Pax4 known to be expressed in pancreatic endocrine progenitors, and NeuroD expressed both in endocrine progenitors and in mature -cells. We also followed the expression of three genes expressed in mature -cells: Pcsk1, Pcsk2, and Abcc8. As shown in Fig. 1K, Nkx6.2 was expressed during the first 3 days of culture, its expression decreasing thereafter. The expression of Ngn3 and Pax4 was turned on and next decreased both with and without mesenchyme, as expected for markers of pancreatic progenitors. The expression of NeuroD was rapidly activated and did not decrease with time, as expected for a gene expressed both in progenitors and mature -cells. Finally, the expression of Pcsk1, Pcsk2, and Abcc8 increased at late time points of in vitro development. Effect of the mesenchyme on the absolute number of -cells. We first compared -cell development from epithelia cultured with or without mesenchyme. In epithelia cultured with or without mesenchyme, the first insulinexpressing 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 both conditions. As shown in Fig. 2K, both with and without DIABETES, VOL. 56, MAY

7 MESENCHYME AND -CELL DIFFERENTIATION FIG. 6. Expression of Ngn3 in embryonic pancreatic epithelia cultured with or without mesenchyme. A K: E13.5 rat pancreatic epithelia cultured for0(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 expression was 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 < Scale bar 50 m. mesenchyme, the number of -cells increased during the first 5 culture days. However, the number of -cells did not increase further from days 5 to 7 without mesenchyme but continued to increase with mesenchyme. As a result, a significantly larger number of -cells developed with than without mesenchyme. Individual -cell size did not vary in the different culture conditions (data not shown). When epithelium was dissected, directly reassociated with mesenchyme, and cultured, its development was similar to the one 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 -cell proliferation. We compared BrdU incorporation in -cells developed without or with the mesenchyme. After 3 days of culture, -cells developed without or with mesenchyme did not incorporate BrdU (Fig. 3A and D). After 5 and 7 days of culture, some -cells developed without mesenchyme incorporated BrdU (Fig. 3B and C); this proliferation was even lower with mesenchyme (Fig. 3E G for quantification). This low proliferation of -cells resembles the one found in the rat embryonic pancreas at E18.5 (Fig. 3H). Thus, the increase in the number of -cells that developed with mesenchyme cannot be explained by an activation of -cell proliferation by the mesenchyme. The mesenchyme activates the proliferation of early pancreatic progenitors. We cultured epithelia for 1 day with or without mesenchyme and added BrdU during the last hour of culture. The percentage of PDX1 cells that incorporated BrdU was 5 2% without mesenchyme (Fig. 4A) and 39 7% with mesenchyme (Fig. 4B, B, and C for quantification). When epithelium was dissected, directly reassociated with mesenchyme, and cultured for 1 day, the proliferation of early PDX1 progenitors was similar to the one of whole pancreas (data not shown). A similar high level of PDX1 -cell proliferation in the presence of mesenchyme can be found in the E13.5 rat embryonic pancreas (Fig. 4D and D ). Thus, the mesenchyme is necessary for maintaining the proliferation of early pancreatic progenitors. The pool of early PDX1 progenitors is maintained in the presence of mesenchyme. To investigate the consequences of proliferation of early PDX1 pancreatic progenitors, we quantified the maintenance of these cells with or without mesenchyme. We cultured pancreatic epithelia with or without mesenchyme and quantified the decrease in the number of early PDX1 /insulin progenitors. At E13.5, the epithelium is composed of early PDX1 /insulin cells (Fig. 5A). In cultures without mesenchyme (Fig DIABETES, VOL. 56, MAY 2007

8 M. ATTALI AND ASSOCIATES 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) or5(b and D) days and pulsed with BrdU during the last hour of culture. Ngn3 expression was detected by in situ hybridization (in blue), whereas BrdU cells were shown in green. Scale bar 25 m. 5B E and J for quantification), the proportion of early PDX1 /insulin progenitors decreased rapidly, and after 3 and 5 days, 41 4 and 23 5% of PDX1 cells remained insulin negative, respectively. With mesenchyme (Fig. 5F I and J for quantification), the decrease in the proportion of early PDX1 /insulin progenitors was slower, and after 3 and 5 days, 76 4 and 51 11% of PDX1 cells remained insulin negative, respectively. By knowing the absolute area occupied by -cells and the percentage of undifferentiated Pdx1 cells, we deduced and compared the absolute number of undifferentiated Pdx1 cells. We found 4.02 and 4.37 times more PDX1 insulin cells with than without mesenchyme after 3 and 5 days of culture, respectively. These results indicate that, in addition to increasing progenitor cell proliferation, the mesenchyme maintains a pool of early PDX1 progenitors. The increase in the number of early PDX1 progenitors by the mesenchyme enhances the -cell pathway. We next monitored Ngn3 expression in epithelia grown with or without mesenchyme. At E13.5, very few cells expressed Ngn3 in rat pancreatic epithelium (Fig. 6A). After 1 day of culture without mesenchyme, Ngn3 expression increased dramatically, reaching a peak followed by a plateau until day 3 then by a sharp decrease on days 5 and 7 (Fig. 6B F). With mesenchyme (Fig. 6G K), the number of Ngn3 cells increased slightly at day 1 compared with epithelia cultured alone, but interestingly, in 3- and 5-dayold cultures, the number of Ngn3 cells was dramatically higher in epithelia cultured with than without mesenchyme (Fig. 6L). In addition, Ngn3 expression was prolonged with mesenchyme: Whereas Ngn3 cells were extremely scarce after 5 days and undetectable after 7 days without mesenchyme, they were present after 5 and 7 days of culture with mesenchyme. Compare Fig. 6D with I, and compare Fig. 6E with J. Quantifications are shown in Fig. 6L. Hypotheses to explain the increase in the number of Ngn3 cells in the presence of mesenchyme include increased differentiation of amplified PDX1 progenitors and proliferation of Ngn3 cells. To test the latter hypothesis, we measured BrdU incorporation by Ngn3 cells grown in vitro from epithelia cultured for 3 or 5 days with or without mesenchyme. Extremely rare Ngn3 cells incorporating BrdU could be found in epithelia that developed either with or without mesenchyme (Fig. 7). This poor in vitro proliferation of Ngn3 cells is reminiscent of the low level of Ngn3 -cell proliferation in the pancreas at E16.5 and E18.5 (data not shown) and could not explain the major increase in the number of Ngn3 cells in the presence of mesenchyme. Thus, our data strongly suggest that the mesenchyme may amplify Ngn3 expression by increasing the magnitude and duration of development of early PDX1 progenitors into Ngn3 progenitors. FGF10 mimics the effects of the mesenchyme on proliferation and differentiation of early PDX1 progenitors. In mice, Fgf10 is expressed in the mesenchyme adjacent to the early pancreatic epithelial buds and required for normal development of the pancreas (17). In rat, FGF10 is expressed in the pancreatic mesenchyme between E12.5 and E18 (27; data not shown). We thus tested whether the effects of the mesenchyme on early progenitor cell proliferation and differentiation could be mimicked by FGF10. Epithelia were cultured for 1 day with or without FGF10. As shown in Fig. 8A C, FGF10 treatment significantly increased the proliferation of PDX1 progenitors. To test the effect of FGF10 on progenitor cell differentiation, epithelia were cultured for 1, 3, or 5 days alone, with mesenchyme, or with FGF10. Without mesenchyme and FGF10, Ngn3 expression increased after 1 day of culture, reached a plateau at day 3, and decreased thereafter. On the other hand, in the presence of either mesenchyme or FGF10, the pattern of expression of Ngn3 was amplified and prolonged (Fig. 8D M). For example, whereas after 5 days of culture, extremely rare Ngn3 cells were detected in epithelia cultured alone (Fig. 8G), Ngn3 cells were abundant in epithelia cultured with FGF10 (Fig. 8M). Such effects of FGF10 on Ngn3 expression were further confirmed by real-time PCR (data not shown). Finally, we quantified the absolute number of -cells developed in epithelia cultured for 5 days in the absence or presence of FGF10. As shown in Fig. 8N P, FGF10 treatment significantly increased the absolute number of -cells that develop. Such effect of FGF10 was also confirmed by real-time PCR (data not shown). DISCUSSION Our results show that the mesenchyme positively regulates the final number of -cells developed from embryonic pancreas. This effect was not due to a direct action of the mesenchyme on the proliferation of either -cells or Ngn3 endocrine progenitors. Instead, the mesenchyme acted by increasing the proliferation of early PDX1 pancreatic progenitors and amplifying and prolonging the formation of Ngn3 endocrine progenitors. We used an in vitro model that allows -cell development from embryonic pancreatic epithelium cultured with and without mesenchyme (28). In earlier work, we showed that -cells developed from embryonic pancreatic epithelium cultured in collagen gel without mesenchyme (20), DIABETES, VOL. 56, MAY

9 MESENCHYME AND -CELL DIFFERENTIATION FIG. 8. FGF10 mimics the effects of the mesenchyme on proliferation and differentiation of early PDX1 progenitor cells. A--C: Pancreatic epithelia were grown without (A) or with (B) FGF10 for 1 day and pulsed with BrdU during the last hour of culture. Immunostaining for PDX1 is in green and BrdU in red. C: Quantification of the number of PDX1 cells in S phase. Data are means SE. **P < 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) mesenchyme or without mesenchyme but with FGF10 (K M). Ngn3 expression was detected by in situ hybridization (in blue). Scale bar 50 m. N P: In vitro effects of FGF10 on -cell development. Pancreatic epithelia were grown without (N) or with (O) FGF10 for 5 days. Insulin cells were revealed in red. Nuclei are stained in blue with Hoechst. P: Quantification of the absolute surface areas occupied by insulin cells that developed during 5 days without or with FGF10. Three rudiments were analyzed for each condition. Data are means SE. **P < Scale bar 50 m. whereas -cells developed poorly when cultured with mesenchyme (21). This inhibitory effect of mesenchyme was due to delayed Ngn3 induction and required a functional Notch pathway (24). A working hypothesis was that hypoxia occurred in collagen gel, activating the Notch pathway and inhibiting endocrine differentiation, a mechanism recently established for other cell types (29). Here, we cultured rat embryonic pancreatic epithelia at the air/liquid interface. Under these conditions, -cells developed in epithelia cultured with or without mesenchyme. -Cells never developed from mesenchyme cultured alone (data not shown). We then found that -cell development in this in vitro model mimicked in vivo development. For example, Ngn3 expression was turned on rapidly then turned off a few days later, in keeping with the reported in vivo pattern (11,30). Also similar to in vivo events, in vitro, Ngn3 induction was followed by the activation of other genes, such as Pax4 and NeuroD, and finally by the induction of markers of terminally differentiated -cells, such as Pcsk1 and Pcsk2, two endoproteolytic enzymes necessary for proinsulin processing, and Abcc8, a sulfonylurea receptor (31 33). It is also interest DIABETES, VOL. 56, MAY 2007

10 M. ATTALI AND ASSOCIATES ing to note that in vitro cell proliferation faithfully replicated in vivo events (17,26,34). Specifically, with mesenchyme, early PDX1 progenitors proliferated at a fast pace. When the cells entered the endocrine pathway and expressed Ngn3, proliferation decreased sharply. Finally, the first -cells that developed after 3 days of culture did not proliferate. This simple in vitro model in which proliferation and differentiation mimic in vivo events is extremely useful for elucidating the effects of the mesenchyme at each step of -cell development. The exact role for the mesenchyme in coordinating progenitor cell proliferation and differentiation is incompletely understood (15,16). Here, we demonstrate that the mesenchyme increases the proliferation of early PDX1 pancreatic progenitors, amplifying and prolonging the formation of Ngn3 endocrine progenitors and ultimately increasing -cell development, and FGF10 mimics the effect of the mesenchyme. However, FGF10 does not provide complete growth of the explants when compared with mesenchyme. We are currently testing whether in addition to FGF10, the mesenchyme generates additional signals that are important for pancreatic epithelial development. We previously demonstrated that FGF10 is produced by embryonic pancreatic mesenchymal cells and is required for the proliferation of early pancreatic progenitors (17). We suggested that failure of the progenitor pool to expand in Fgf10 mutants might lead to a reduced number of progenitors available for differentiation, ultimately resulting in an inadequate number of cells to produce a normal pancreas. However, the detrimental effects of FGF10 deletion hindered the interpretation of the role for FGF10 in later stages of development (17). However, in transgenic mice ectopically expressing FGF10 in the pancreatic epithelium, epithelial cell proliferation increased but pancreatic differentiation of all cell types was markedly decreased (18,19). Such a phenotype was also observed when E10 mouse pancreatic epithelium was cultured in matrigel in the presence of FGF10 (35). This inhibition of differentiation was unexpected, and the contradiction between the phenotype of FGF10-deficient mice and transgenic mice with ectopic FGF10 expression or E10 mouse pancreatic epithelium grown with FGF10 was unexplained. For example, the phenotype of FGF10-deficient mice suggested that FGF10 did not inhibit endocrine cell differentiation. In the absence of FGF10, there was no excessive expression of early markers for endocrine cells, such as Isl1 and Ngn3. Moreover, glucagon-expressing cells did not form prematurely or in excess (17). In contrast, endocrine cell differentiation was strongly inhibited in transgenic mice overexpressing FGF10 or in E10 mouse pancreatic epithelium grown with FGF10 (18,19,35). One hypothesis for explaining these apparent contradictions involves the timing of FGF10 expression in transgenic mice. In these mice, FGF10 expression is controlled by the Pdx1 promoter, which is expressed very early in the pancreas, before the mesenchyme condenses around the gut tube. At this early stage, FGF10 treatment of dorsal pancreatic endoderm explants was sufficient to increase the expression of Ptf1a, a transcription factor needed to complete pancreatic specification (36,37). Thus, one possibility is that early FGF10 misexpression in the pancreas modifies the pancreatic cell-differentiation program, a mechanism recently suggested to explain the pancreatic phenotype of mice that overexpress Wnt1 under the control of the Pdx1 promoter (38). These mice have pancreatic agenesis. Comparisons of their phenotype with that of mice deficient in -catenin suggest that Wnt1 overexpression at early development stages may reflect alternative roles for Wnt signaling, such as specifying the fate of the intestine (39). Difference in the stage of development of the explants could also explain the differences 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 also explain the differences. Although, here, we grew explants on filters at the air/liquid interface, Miralles et al. performed cultures in matrigel that contains basement membrane proteins that are not fully characterized. Taken together, our data indicate that 1) the pancreatic mesenchyme increases the final -cell population by activating the proliferation of early PDX1 pancreatic progenitors; and 2) the effects of the mesenchyme on proliferation and differentiation can be mimicked by FGF10. We propose that early PDX1 pancreatic progenitors represent an expansion pool characterized by a significant proliferative potential that should be exploited for expansion with FGF10 and differentiation into -cells. Interestingly, Baetge and colleagues (40) recently directed differentiation of human embryonic stem cells into insulinproducing cells by mimicking embryonic development. However, as noted by the authors, the protocol must be further improved to produce therapeutic -cells. The information we provide here should be useful to define the exact timing during which FGF10 should be used to amplify pancreatic progenitors and thus increase the final number of -cells that will develop. ACKNOWLEDGMENTS M.A. received support from the French Ministry for Research and Technology and from the Association Pour la Recherche Médical. 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