Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development

Developmental Biology 361 (2012) 277–285

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Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development Anthony Beucher a, Mercè Martín a, Caroline Spenle b, Martine Poulet a, Caitlin Collin a, Gérard Gradwohl a,⁎ a Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR 7104; Université de Strasbourg (UdS), 67404 Illkirch, France b Unité mixte de Recherche INSERM-UdS 682, 67200 Strasbourg, France

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Article history: Received for publication 6 July 2011 Revised 8 October 2011 Accepted 10 October 2011 Available online 26 October 2011 Keywords: Pancreas Endocrine progenitors Cell fate Plasticity Neurog3 Ngn3 Diabetes

a b s t r a c t During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to nonendocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3+ progenitors develop into hormone-expressing cells in a fast process taking less then 10 h. Furthermore, fate-mapping studies in heterozygote (Neurog3CreERT/+) and Neurog3-deficient (Neurog3CreERT/CreERT) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities. © 2011 Elsevier Inc. All rights reserved.

Introduction During pancreas development it is thought that all differentiated cells arise from a pool of multipotential progenitor cells which, through successive cell fate choices, give rise to the exocrine (acinar and ductal) and endocrine (islet) lineages. Characterization of the differentiation potential and lineage relationship of these pancreatic progenitors throughout embryogenesis is essential to understand islet cell development and might be relevant for the generation of therapeutic beta cells from human embryonic stem cells. In the mouse embryo, during the initial phase of pancreas development (E9.5–E10.5), the pancreatic epithelium is composed of a cluster of unorganized endodermal progenitor cells expressing markers such as the transcription factors Pdx1, Ptf1a, Sox9, HNF1β or Nkx6.1 (Kawaguchi et al., 2002; Lynn et al., 2007; Maestro et al., 2003; Offield et al., 1996; Sander et al., 2000; Seymour et al., 2007; Solar et al., 2009). Genetic lineage tracing

⁎ Corresponding author at: Institute of Genetics and Molecular and Cellular Biology, Development and Stem Cell Program, 1 rue Laurent Fries, F-67404 Illkirch, France. Fax: + 33 3 9065 3201. E-mail address: [email protected] (G. Gradwohl). 0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.10.025

studies, based on the use of transgenic mice expressing the Cre recombinase under the control of the regulatory elements of some of these transcription factors, demonstrated that these early pancreatic progenitor cells can develop into exocrine cells (acinar and ductal) as well as into the different endocrine cellular subtypes including insulinproducing β cells (Gu et al., 2002; Kawaguchi et al., 2002; Kopp et al., 2011; Solar et al., 2009). From about E12 onwards, branching of the pancreatic epithelium within the surrounding mesenchyme starts and progenitors are then compartmentalized in the so-called tip and trunk domains (Zhou et al., 2007). Zhou and colleagues suggested that after E14.5, endocrine cells arise exclusively from precursors located in the embryonic duct epithelium in the trunk. Recent temporal genetic fate mapping studies of embryonic trunk cells marked by the transcription factors Hnf1β or Sox9 gave some additional information on the cellular origin of pancreatic endocrine and exocrine cells. Thus, after E13.5 Hnf1β trunk cells can give rise to ductal and endocrine cells but have lost their capacity to differentiate into acinar cells (Solar et al., 2009). In contrast, the Sox9-domain remains multipotent until birth and thus keeps the competence to differentiate into acinar cells which arise from a subpopulation of Sox9 cells located at the extremity of the embryonic trunks (Kopp et al., 2011). These observations suggest that even tough they express the same marker,

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pancreatic progenitors are a heterogeneous domain of cell, which have different potentialities depending their localization and developmental stage. It is acknowledged that the expression of the basic helix loop helix (bHLH) transcription factor Neurogenin3 (Neurog3) implements the endocrine program in multipotent progenitors based on the following findings. First, inactivation of Neurog3 precludes the development of all islet cells (Gradwohl et al., 2000; Heller et al., 2005; Lee et al., 2002). Of note, residual glucagon expressing cells have been reported before E12.5 in one Neurog3 knock out line (Wang et al., 2008). Second, forced expression of Neurog3 in Pdx1-positive progenitors results in premature endocrine differentiation (Apelqvist et al., 1999; Johansson et al., 2007; Schwitzgebel et al., 2000). Interestingly the competence of these pancreatic progenitors to contribute to the different endocrine cell types upon Neurog3 stimuli varies during embryogenesis (Johansson et al., 2007). Third, lineage tracing experiments demonstrated that all endocrine cells derive from Neurog3-expressing progenitor cells (Gu et al., 2002; Heller et al., 2005; Schonhoff et al., 2004). Endocrine committed Neurog3 + cells are essentially post-mitotic (Miyatsuka et al., 2011) and in vivo clonal analysis support that they are unipotent, meaning that a single Neurog3 + cell gives rise to a single islet cell-type (Desgraz and Herrera, 2009). The later implies that although they express the same marker (Neurog3 +) islet progenitor cells are a heterogeneous population of cells. Neurog3 thus marks a population of progenitors committed to the endocrine lineage that can be found initially as scattered cells localized in the pancreatic bud and later in the trunk epithelium. Despite the central role of Neurog3 in the specification of pancreatic islets, recent studies suggest that Neurog3-positive progenitors might eventually adopt alternative fates (Schonhoff et al., 2004; Wang et al., 2009b). Furthermore, it appeared that Neurog3 expression has to reach a certain threshold to consolidate the endocrine fate (Wang et al., 2009b). The latter conclusion has been drawn by Wang and colleagues using a lineage tracing approach based on the expression of constitutive Cre recombinase in Neurog3expressing cells. They showed that in total absence of Neurog3 and to a lower extent in low Neurog3 dosage, Neurog3-induced progenitors adopt an acinar or ductal destiny. Thus, these data imply that endocrine progenitors arise from multi or at least bipotent precursors. The identity and potentiality of such precursor cells, lying immediately upstream of Neurog3-positive progenitor cells, are still elusive. It is also unclear whether the competence of Neurog3expressing cells to give rise to non-endocrine lineages depends on the developmental stage. In this study our aim was to explore the plasticity of Neurog3induced cells throughout development in heterozygous and more particularly in Neurog3-deficient conditions. To address this question we used an inducible Cre/lox lineage tracing approach. We took advantage of the Neurog3 CreERT/+ mice (Wang et al., 2009a) where cells transcribing Neurog3 gene can be genetically marked at a specific developmental time point and the ability of the tagged progenitor to contribute to a specific lineage determined at later stages. Importantly, this mouse model also allows the identification and tracking of Neurog3-induced progenitors in Neurog3 homozygous null mutant mice. Thus, cells which have been instructed to turn on the Neurog3 gene but cannot adopt an endocrine destiny because they fail to express Neurog3 transcription factor can be labeled and studied. Our hypothesis was that information on the alternative destiny of these Neurog3-induced cells during pancreas development would provide important insight on the competence/plasticity of the failed endocrine progenitor. Using this mouse model, we show that islet cell progenitors differentiation into hormone-producing cells is a fast process leading to hormone expression within 10 h. In absence of Neurog3 expression, after E12.5 Neurog3-induced cells do not delaminate and differentiate

into ductal cells, whereas before E12.5 Neurog3-induced cells differentiate into acinar or ductal cells. Neurog3-induced cells thus lose their acinar potential during development. These results suggest that islet progenitor cells arise from multipotent precursors in the pancreatic bud. However starting from E12.5, endocrine cells arise from bipotent precursor cells located in the trunk already committed to the ductal/endocrine lineage. Furthermore these data strongly support the bipotent nature of trunk precursor cells rather then being individual precursors with a ductal or endocrine potentiality. Results Neurog3-induced cells differentiate into hormone-expressing cells in 10 h in mouse embryos To characterize the biology and address the potential of Neurog3induced cells at different developmental stages, we used Neurog3 CreERT knock-in mice (Wang et al., 2008). We first characterized the accuracy of this model for temporal lineage tracing studies. In Neurog3 CreERT/+ mice, the Tamoxifen inducible CreER™ recombinase (hereafter referred to as CreERT) replaces the Neurog3 coding sequence (Fig. 1a). By mating these mice with the Cre reporter mice R26RlacZ (Soriano, 1999) or R26ReYFP (Srinivas et al., 2001), a pulse of Tamoxifen allows the genetic labeling of Neurog3-induced cells and their progeny by constitutive expression of the β-galactosidase (β-gal) or the enhanced yellow fluorescent protein (eYFP). Furthermore, by generating Neurog3CreERT/CreERT homozygous mice, it is possible to genetically tag and follow the alternative fate of Neurog3-induced cells, which do not express the proendocrine transcription factor. Pancreatic endocrine progenitor cells are characterized by the transient expression of Neurog3. During their differentiation into the various islet cell types, Neurog3 expression is turned off before the onset of hormone expression. Although, the knock-in approach ensures a faithful expression of the CreERT in Neurog3 + cells, differences in stability between Neurog3 and CreERT proteins could result in misinterpretation of the identity of the labeled cells (e.g. labeling of post-Neurog3 cells if CreERT is more stable than Neurog3). We thus first verified the overlapping expression of CreERT and Neurog3 proteins on E15.5 pancreas sections. As expected, cytoplasmic CreERT expression is detected in almost all Neurog3 + cells, and Neurog3 −/ Cre + cells were rarely observed (Fig. 1b). To genetically label Neurog3-induced cells at a precise time point, Neurog3 CreERT/+ females were mated with R26R eYFP males and injected with a single dose (1 mg) of 4-hydroxy-Tamoxifen (4-OH-TM) at E15.5. Nuclear translocation and robust expression of CreERT in Neurog3 + cells were observed after 4-hydroxy-Tamoxifen (4-OH-TM) injection (Fig. 1c), which advocates that recombination will very likely occur in Neurog3 + cells. The presence of some CreERT +/Neurog3 − nuclei (Fig. 1c) suggests that CreERT perdures after Neurog3 is turned off. Importantly, we never observed CreERT expression in hormonepositive cells in uninduced or 4-OH-TM treated embryos (Fig. 1d, e). Previous publications reported that Tamoxifen activated CreERT is translocated in the nucleus and recombination initiated within 6 h (Ahn and Joyner, 2004) (Hayashi and McMahon, 2002). In agreement with these findings, eYFP-labeled cells can already be detected in the pancreas 10 h after 4-OH-TM injection (Fig. 2b), demonstrating that recombination is effective in this time frame (shorter period were not tested). Two populations of eYFP-tagged cells can be observed 10 h after 4-OH-TM injection: eYFP +/Neurog3 + cells, which are endocrine progenitor cells and eYFP +/Neurog3 −, likely corresponding to differentiating endocrine cells. Importantly, 24 h after 4-OH-TM injection, most of eYFP + cells no longer express Neurog3 (97.1 ± 0.57%, Fig. 2c), but express endocrine transcription factors e.g. Insm1, MafB and MafA (Fig. S1c), demonstrating that these eYFP +/Neurog3 − cells are differentiating post-Neurog3 endocrine cells. To determine

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Fig. 1. Temporal lineage tracing with Neurog3CreERT mouse. (a) Schematic of the Neurog3CreERT Knock-in and Cre reporter alleles and mating scheme. A Tamoxifen-inducible Cre recombinase (CreER™) expressed specifically from the Neurog3 locus removes the “STOP” cassette in the Rosa26 locus upon Tamoxifen treatment, allowing constitutive expression of the β-gal or the eYFP. (b–e) Representative confocal images showing immunofluorescence for Neurog3, Cre and islet hormones on cryosections of E15.5 (b–d) or E16.5 (e) Neurog3CreERT/+ pancreas, untreated (b, d) or 24 h after 4-OH-Tamoxifen (4-OH-TM) treatment (c, e). (b–c) Nuclear translocation of CreERT is activated in Neurog3+ cells upon 4-OH-TM injection. (d–e) The CreERT recombinase is not expressed in hormone-producing cells. Arrows point to double stained cells. *, this cell does not stain for DAPI (not shown), thus the optical section does not cross through the nucleus and the red staining corresponds to cytoplasmic CreERT (b–c) Scale bar 20 μm. (d–e) Scale bar 50 μm.

the time required for the differentiation of a hormone-producing cell from a Neurog3 + progenitor, we analyzed Insulin, Glucagon and Pancreatic Polypeptide (PP) expression in the Neurog3 + cells progeny. 24 h

and even 10 h after 4-OH-TM administration, all these hormones are commonly expressed in eYFP+ cells (Fig. 2d–i). At 10 h, 89.5% of eYFP+ cells coexpress Insm1 while 4.5% are hormone-positive (Fig.

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Fig. 2. Endocrine subtype specification in 10 h. (a) Schematic of the lineage tracing experiment. (b–i) Confocal immunofluorescence images of Neurog3CreERT/+;R26ReYFP embryonic pancreas, 10 h (b, d–f) and 24 h (c, g–i) after 4-OH-TM injection. Neurog3 is still expressed in some eYFP+ cells 10 h (b, arrows), but not 24 h after injection (c) demonstrating that Neurog3 is expressed less than 24 h in endocrine progenitors. Insulin (d), Glucagon (e, arrow) and PP (f) hormones are detected in Neurog3 descendants 10 h after the labeling of Neurog3 expressing cells. (j) Kinetic of an endocrine progenitor cell differentiation. (b–c) Scale bar 50 μm. (d–i) Scale bar 10 μm.

S1d) suggesting that the later derive from Neurog3+ cells where recombination occurred just before Neurog3 is turned down and that differentiation does not occur in less then 10 h. Taken together our data

indicate that endocrine differentiation occurs with an initial transient expression of Neurog3, which lasts less than 24 h, followed within 10 h by hormones synthesis (Fig. 2j).

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Neurog3-induced cells fail to delaminate in Neurog3-deficient mice resulting in abnormal morphogenesis of the trunk epithelium To gain insight into the potentiality of Neurog3-deficient cells we first characterized the fate of the Neurog3-induced cells in Neurog3 CreERT/CreERT; R26R lacZ embryos at E15.5, when numerous Neurog3-positive cells are found in the trunk epithelium. Pregnant mice were injected with 4-OH-TM at E15.5 and embryos were harvested one day later (Fig. 3a). The overall number of labeled cells varied greatly among the embryos, likely due to variation in the availability/efficacy of 4-OH-TM. However, we did not observe a statistically significant difference between the number of genetically labeled cells between heterozygous and Neurog3-deficient embryos (Fig. S4). At E16.5, we detected X-gal+ cells within the trunk epithelium of Neurog3CreERT/+; R26RlacZ control and Neurog3CreERT/CreERT; R26R lacZ embryos (Fig. 3a–f). In the control pancreas, 24 h after 4-OH-TM injection, X-gal+ cells have started to delaminate from the trunk epithelium (Fig. 3c, e). In contrast, in Neurog3-deficient pancreas, labeled cells

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(X-gal+) are stuck in the trunk (Fig. 3d, f). Delamination failure of labeled cells in absence of Neurog3 transcription factor are even more obvious 48 h after 4-OH-TM injection (Fig. 4 compare h and i). These results demonstrate that Neurog3 is necessary for the delamination of developing islet cells from the pancreatic epithelium. Because Neurog3 activated cells have not delaminated in the mutants and still expressed Pdx1 (Fig. 3d, f) we postulated that these cells could maintain a proliferative capacity. Indeed, in the control pancreas, most if not all of the CreERT + cells are Ki-67− (Fig. 3g), indicating that endocrine progenitors are mainly post-mitotic. In contrast, in the absence of Neurog3 expression, almost all the CreERT + cells where Neurog3 transcription has been activated are dividing (Fig. 3h) in agreement with a recent report showing the importance of Neurog3 in the regulation of cell cycle exit (Miyatsuka et al., 2011). Accumulation of such dividing cells that cannot delaminate and engage an endocrinogenic differentiation program resulted into abnormal morphogenesis of the trunk epithelium with cystic like structures (Fig. 3f and data not shown). Thus these findings demonstrate that Neurog3 is induced in proliferating precursors before cell cycle exit and delamination. Neurog3-induced cells have the potential to develop into ductal cells throughout development in Neurog3-deficient mice We next determined the differentiation potentiality of Neurog3induced cells in Neurog3-deficient mice throughout pancreas development. Neurog3CreERT/+ and Neurog3CreER/T ; R26ReYFP mice were crossed and pregnant females injected with a single dose of 4-OH-TM at E10.5, E11.5, E12.5 and E15.5 (Figs. 4 and 5). Embryos were essentially analyzed at E17.5. When probing the endocrine lineage we showed that from E10.5 to E12.5 Neurog3+ cells can be efficiently labeled and give rise to α-cells, or at later stages to β-cells in heterozygous mice (Fig. 5a, c, e, g) in agreement with previous studies (Johansson et al., 2007). As expected endocrine cells did not develop in Neurog3CreERT/CreERT; R26ReYFP embryos (Fig. 5b, d, f and not shown). We first addressed the ductal potentiality of Neurog3-deficient cells. Consistently with the data described above, using the R26RLacZ reporter, in control embryos (Neurog3CreERT/+; R26ReYFP), Neurog3+ cell descendants (EYFP+) have delaminated from the trunk epithelium to form islets (Fig. 4h). In contrast, in absence of Neurog3 (Neurog3CreERT/CreERT; R26ReYFP), the progeny of Neurog3-induced cells is still located in the trunk and expresses Muc1 and Sox9, two duct cell markers at this late embryonic stage (Fig. 4i, S2g). When labeled at E15.5, most of the eYFP+ cells progressively lose Pdx1 expression in Neurog3-deficient embryos (Fig. S2j, l) further supporting that the tagged pancreatic progenitors differentiate into duct cells. Importantly, ductal destiny was observed for Neurog3-induced cells in KO mice independently of the embryonic stage when progenitors have been genetically labeled (Fig. 4c, e, g). In heterozygous mice, Neurog3-induced cells can differentiate into duct cells essentially before E12.5 but very rarely after (Fig. 4b, c and S2b, c and not shown). Theses results demonstrate that in Neurog3-deficient mice, Neurog3-induced cells can give rise to ductal cells during the first and secondary transition period. Neurog3-induced cells differentiate into acinar cells only before E12.5

Fig. 3. Neurog3-induced progenitors divide and do not migrate out of the pancreatic epithelium in Neurog3-deficient mice. (a–b) Whole mount X-gal staining of β-Gal expressing cells in E16.5 pancreas, 24 h after 4-OH-TM injection. (c–f) X-gal staining (blue) and immunohistochemistry for Pdx1 (brown). e and f are higher magnifications of boxed area in c and d respectively. Neurog3-mutant cells (d, f, blue cells) do not delaminate from the epithelium in contrast to the progeny of Neurog3CreERT/+ control cells (c, e, arrows) resulting into cystic like epithelial structures (d, f). (g–h) Confocal immunofluorescence images of heterozygous and Neurog3-mutant E16.5 pancreas sections immunostained with anti-Ki67 and anti-Cre antibodies, 10 h after 4-OH-TM injection. Whereas the Neurog3+ (CreERT) cells are mainly post-mitotic (g), most of the Neurog3-mutant cells (CreERT) expressed the proliferative marker Ki-67 (h). Bottom panels are higher magnifications of the boxed area in g and h. Scale bar 50 μm.

Previous lineage tracing of Neurog3+ cells in transgenic mice have reported that a small fraction of acinar cells can arise from Neurog3+ cells (Gu et al., 2002; Schonhoff et al., 2004; Wang et al., 2009b). Here we examined the acinar potentiality of Neurog3-induced cells at various developmental stages. In absence of Neurog3, when pregnant females were injected with 4-OH-TM at E10.5 or E11.5, Neurog3-induced cells differentiate into many amylase-positive acinar cells (89.6± 1.67% of eYFP cells are acinar cells when injected at E11.5 in Neurog3 CreERT/CreERT; eYFP R26R mice, Fig. 5b, d). Importantly further experiments revealed that by E12.5, Neurog3-deficient cells lost this competence to differentiate into acinar cells (Fig. 5f, h). When the same analysis was performed at

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progenitors born before E12.5 (Fig. S3 compare a and b). Interestingly, we noticed that when Neurog3-induced cells were labeled at E10.5 or E11.5 (but not at E12.5) in Neurog3-deficient embryos, numerous clustered eYFP-labeled cells were subsequently found at E17.5 (Fig. 5 compare b, d to f and S3 compare b to d), suggesting a clonal origin of YFP+ acinar cells. Labeled acinar clusters were also found in control Neurog3CreERT/+ embryos in similar experiments but much fewer cells were labeled (Fig. 5a’ and S3a). Further supporting a clonal origin, analysis of pancreas at E12.5 two days after injection, revealed only isolated eYFP+ cells (Fig. 5k–n). We thus postulated that clusters of acinar cells arose from single Neurog3-expressing cells, which adopted an acinar fate and later proliferated. Taken together, our data demonstrate that during the primary transition, Neurog3-induced cells can give rise to acinar cells. However, at E12.5 and thereafter, when Neurog3-induced cells are located in the trunk domain of the branching epithelium, they loose their capacity to differentiate into the acinar lineage. Discussion During pancreas embryogenesis, all hormone-expressing endocrine cells differentiate from endocrine progenitor cells marked by the transcription factor Neurog3, which is strictly required for endocrine commitment. However, the plasticity and the biology of Neurog3-induced cells during development remain incompletely characterized. Therefore, we took advantage of a mouse model where Neurog3-induced cells can be genetically labeled at different time points to address these questions. Our experiments first revealed a time window of about 34 h for islet progenitor cell differentiation in vivo. Second, we demonstrate that Neurog3 is required for delamination of the developing islet cells. Third, we show that Neurog3induced cells have different potentiality during pancreas development (Fig. 6). Importantly, we provide evidence that after the secondary transition islet progenitor cells arise through truly bipotent duct/endocrine progenitor cells. Kinetic of endocrine differentiation

Fig. 4. Reallocation of Neurog3-induced progenitor cells into ductal cells in Neurog3-deficient mice throughout development. (a) Schematic of the lineage tracing experiment. (b–i) Confocal immunofluorescence images of E17.5 pancreas sections from heterozygous (Neurog3CreERT/+ ; R26ReYFP), or Neurog3-deficicient mice (Neurog3CreERT/CreERT; R26ReYFP/+) treated with 4-OH-TM at various time points between E10.5 and E15.5. Neurog3-induced progeny differentiate into Muc1+ duct cells at every stage analyzed in Neurog3-deficient mice (c, e, g, i) whereas Neurog3-induced cells differentiate into Muc1+ cells only before E12.5 in heterozygotes (b, d). Arrows point to eYFP+/Muc1+ cells. (b–i) Scale bar 20 μm.

Using temporal lineage tracing, we have determined that Neurog3 protein expression lasts less than 24 h in pancreatic endocrine progenitors. Insulin, glucagon or PP peptides are detected only 10 h after Neurog3 expression is turned off. Previous studies based on data obtained with a Neurog3-timer mice suggest that Neurog3 was transcribed for about 6 h in the endocrine progenitors, and that it takes another 12 h for these cells to express insulin or glucagon transcripts (Miyatsuka et al., 2009). In the later study, differentiation was analyzed in dissociated islet progenitor cells maintained in culture. Variation in the time window for Neurog3 expression and hormone synthesis between both studies might thus result from differences between mRNA (Miyatsuka et al., 2009) and protein half lives (this study). Alternatively culture conditions might accelerate the development of islet cells. Taken together our data imply that, starting from the beginning of Neurog3 expression, it takes less than 34 h for the generation of a hormone-producing cell in the mouse embryo. Neurog3 is necessary for the migration of developing islet cells

E19.5, 4 days after injection with 4-OH-TM, we similarly did not detect any labeled acinar cell (Fig. 5i, j). Tracing of Neurog3 heterozygous cells labeled between E10.5–E11.5 showed that they could similarly differentiate into acinar (Fig. 5a’) cell lineage. However, such a non-endocrine destiny for a Neurog3 + cells in heterozygotes embryos was a rare event in our experimental system compared to homozygotes. These findings are consistent with previous data suggesting that reduced production of Neurog3 promotes acinar cell differentiation (Wang et al., 2009b). Importantly in heterozygous cells, similarly to Neurog3deficient cells, the acinar fate was restricted to Neurog3-induced

In the embryonic pancreas, endocrine progenitors arise within the pancreatic epithelium. Developing endocrine cells subsequently delaminate from the epithelium and migrate into the mesenchyme where they aggregate into clusters, the islets of Langerhans. Our data clearly demonstrate that this process is strictly dependent on Neurog3. Indeed in absence of Neurog3, we showed that failed endocrine progenitors do not migrate from the pancreatic epithelium, ultimately leading to perturbed trunk morphogenesis. In agreement with our findings, Magenheim et al. (2011) described recently reduced branching and enlarged pancreatic duct-like structures in Neurog3-

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Fig. 5. Neurog3-induced cells can give rise to acinar cells only before E12.5. Confocal immunofluorescence images of heterozygous and Neurog3-mutant embryonic pancreas sections at E12.5, E17.5 or E19.5, from pregnant mice injected with 4-OH-TM between E10.5 and E15.5. (a–c): E10.5 and E11.5 Neurog3+ progenitors give rise essentially to endocrine cells (a, c) and rarely differentiate into Amylase+ acinar cells (a’), in contrast to Neurog3-deficient mice where many Neurog3-induced cells differentiate into acinar cells (b, d). From E12.5 Neurog3+ progenitors differentiate only into endocrine cells (e, g) and Neurog3-deficient cells have lost their competence to differentiate into acinar cells (f, h, j). (k–n) At E10.5, Neurog3-induced cells can differentiate into Cpa1+ cells in heterozygous and Neurog3 deficient mice. (m, n) Higher magnification of insets in k’ and l. Yellow and blue arrows point to eYFP labeled acinar and endocrine cells respectively. Red arrows point to β-gal expressing cells. Scale bar 20 μm.

deficient mice that could result from delamination failure. Consistently, it has been demonstrated, that Neurog3 is sufficient to trigger cell delamination from the chicken endoderm (Gouzi et al., 2011; Rosenberg et al., 2010). Interestingly Gouzi et al. (2011) provide some evidence that Neurog3 induces an Epithelial-to Mesenchymal Transition (EMT), by regulating at the post-transcriptional the level, the expression of the transcription factor Snail2, known to trigger EMT (Nieto, 2009). Thus, Neurog3 is both necessary and sufficient to trigger the delamination of developing endocrine progenitors from the pancreatic epithelium. During the secondary transition islet cells arise from bipotent duct/endocrine progenitor cells and not from domain of cells having distinct potentialities Recent lineage tracing experiments demonstrated that during the secondary transition, Hnf1β + early duct cells (also called embryonic cords) differentiate exclusively into duct or endocrine cells (Solar et al., 2009). From these results, it is impossible to conclude whether the endocrine and ductal lineages arise from a common bipotent progenitor or from distinct progenitors expressing Hnf1β +. In the same line, although Hnf1β and Sox9 expressions overlap extensively in

the embryonic cords, it has been reported that Sox9 + cells can give rise to acinar cells (in addition to endocrine and duct cells) until birth, likely from terminal duct progenitors which do not express Hnf1β. Thus these temporal cell-tracing experiments revealed that domains of progenitor cells of the early duct epithelium, and expressing a specific maker, differ in their developmental potential. At the single cell level however there is no formal demonstration that a given Hnf1β or Sox9 + progenitor is bi- or multi-potent. Here we providence evidence that after progenitor cell compartmentalization into tip and trunk domains (around E12.5) and in absence of Neurog3 expression, Neurog3-induced trunk/cord cells differentiate solely into duct cells. In control experiments performed in heterozygotes, the vast majority if not all Neurog3 + cells adopt an endocrine destiny. Thus these data strongly support the hypothesis that islet progenitors indeed arise from bipotent duct/endocrine precursors through a binary cell fate decision during the secondary transition. Interestingly before E12.5, Neurog3-induced cells still have the potential to differentiate into acinar cells (as well as duct cells). This implies that during pancreas development, the first endocrine cells differentiate from multipotent acinar/duct/endocrine progenitors or at least from bipotent endocrine/acinar or endocrine/duct progenitors. After

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Fig. 6. Summary of the lineage tracing experiments defining the destiny of Neurog3-induced cells in heterozygous and Neurog3-deficient mice. A, Acinar cell; D, duct cell; E, endocrine cell. Full line: main differentiation ways. Dashed line: rare events.

Recent strategies to generate therapeutic beta cells from embryonic or induced pluripotent stem cells are essentially based on the recapitulation of embryonic differentiation programs. Using this approach, immature beta cells have been obtained from human embryonic stem cells (D'Amour et al., 2006). It is not known whether the kinetic of differentiation influences the functionality of the insulin-expressing cells obtained. Similarly the importance of the cellular mechanism underlying beta cell generation has not been studied. Our data suggest that during pancreas development islet progenitors can be generated from progenitors with distinct potentialities. Thus do hES-derived Neurog3+ cells derive from multipotent progenitors or bipotent endocrine/ductal progenitors? It is tempting to speculate that hES-derived insulin-expressing cells resemble the immature endocrine cells generated in the early pancreatic bud from metastable Neurog3+ cells originating from multipotent progenitors. Therefore, novel strategies could focus on mimicking the generation of beta cells from bipotent endocrine/ductal trunk progenitors occurring during the secondary transition. Recently pancreatic tubulogenesis has been suggested to promote exposition of trunk progenitors to a specific microenvironment important for appropriate cell fate determination (Kesavan et al., 2009). Identification of the conditions to reproduce such a niche might be the key for the generation of bipotent endocrine/ductal progenitors and the subsequent differentiation of functional beta cells in vitro. Material and methods

branching is initiated at E12.5, endocrine cells are then generated exclusively through bipotent duct/endocrine progenitors. The competence of Neurog3 heterozygous cells to give rise to non-endocrine cells is restricted to the primary transition period Previous studies provide evidence that during pancreas development, Neurog3 gene dosage controls endocrine versus exocrine cell fate destiny in Neurog3-expressing cells (Wang et al., 2009b). This conclusion was based on the observation that mice heterozygous for Neurog3 have a reduced production of Neurog3 protein as well as a higher number of Neurog3 + cells giving rise to acinar as well as ductal cells compared to wild-type. Therefore, only cells expressing sufficiently high levels of Neurog3 would differentiate into endocrine cells. The current temporal lineage tracing study revealed that Neurog3-positive cells, in Neurog3 CreERT/+ mice, are only competent to differentiate into acinar or duct cells before E12.5. We only exceptionally observed rare duct cells and never any acinar cell arising from Neurog3-expressing cells after this stage. This implies that Neurog3 gene dosage operates to regulate endocrine versus non-endocrine cell allocation in competent cells present during the primary transition period. Thus, cells born before E12.5 and unable to reach this Neurog3 threshold would be at the origin of exocrine cells arising from Neurog3-expressing cells observed in this study and in constitutive lineage tracing experiments that have used Neurog3 driven Cre transgenic mice (Cras-Meneur et al., 2009; Gu et al., 2002; Schonhoff et al., 2004; Wang et al., 2009b). In line with these observations, deletion of HNF6 in endocrine progenitors also increases allocation of progenitor cells to the exocrine lineage (Zhang et al., 2009). As HNF6 is known to activate transcription of Neurog3 (Jacquemin et al., 2000), HNF6 function could be to sustain Neurog3 expression and promote endocrine destiny. Thus Neurog3-induced cells have certain plasticity before E12.5 and can give rise to endocrine and eventually acinar or duct cells, whereas after this stage they are strictly committed to the endocrine lineage. In good agreement with our findings it is also from E12.5 that an antagonistic repressive activity of Nkx6 and Ptf1a transcription factors has been suggested to restrict multipotent pancreatic progenitors to ductal/endocrine versus acinar fates (Schaffer et al., 2010). Thus, Nkx6.1 and 6.2 could determine competent bipotent duct/endocrine progenitors that will differentiate into endocrine cell upon Neurog3 expression.

Mouse strains and tamoxifen treatment The Neurog3CreERT/+ strain was described previously (Wang et al., 2008). Neurog3CreERT/+ females were mated with Neurog3CreERT/+; R26ReYFP/eYFP or Neurog3CreERT/+; R26RlacZ/lacZ males in order to generate mutant embryos Neurog3CreERT/CreERT; R26ReYFP/+ or Neurog3CreERT/CreERT; R26RlacZ/+. The noon of the day of a vaginal plug was designated as E0.5. R26RlacZ/+ and R26ReYFP/+ mouse lines were generous gifts from P. Soriano and F. Costantini. Pregnant females were injected intraperitoneally with 1 mg 4-hydroxy-Tamoxifen (4-OH-TM) at indicated embryonic stages. 4-OH-TM (H-6278 Sigma) was prepared at 10 mg/mL in 10% ethanol and 90% corn oil. β-Galactosidase activity detection For 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal) staining, tissues were fixed for 30 min at room temperature in 0.2% glutaraldehyde, 5 mM EGTA, 2 mM MgCl2 in 0.1 M sodium phosphate pH 7.3, washed three times for 10 min in LacZ wash buffer (2 mM MgCl2 in 0.1 M sodium phosphate pH 7.3, 0.02% NP-40) and whole mount staining was performed in a solution containing 1 mg/ml X-Gal, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide in LacZ wash buffer at 37 °C for 4 h. After staining, samples were washed in PBS, post-fixed with Paraformaldehyde (PFA) 4% at 4 °C and processed for wax sections. Immunohistochemistry Digestive tracts were fixed in PFA 4%, o/n at 4 °C, washed in PBS, equilibrated in 20% sucrose at 4 °C and embedded in OCT compound. Immunohistochemistry analyses were performed on 10 to 14 μm cryosections. The following primary antibodies were used: guinea pig antiNeurog3 at 1:1000 (provided by M. Sander, University of California — San Diego, La Jolla, CA, USA), rabbit anti-Cre at 1:2000 (Eurogentec Covance), rabbit anti-GFP at 1:500 (Molecular Probe, A-6455), rat anti-Ecadherin at 1:200 (Invitrogen), guinea pig anti-Insulin at 1:1000 (Linco), guinea pig anti-Glucagon at 1:2000 (Linco), guinea pig antiPP at 1:1000 (Linco), mouse anti-Ki-67 at 1:100 (Novo castra), Armenian hamster anti-Muc1 at 1:500 (Thermo), goat anti-Sox9 at 1:50

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(Santa Cruz), guinea pig anti-Pdx1 at 1:500 (provided by C. Wright, Vanderbilt University, Nashville, TN, USA), goat anti-Amylase at 1:200 (Santa Cruz), rabbit anti-Amylase at 1:1000 (Sigma), chicken anti-βgalactosidase at 1:5000 (Abcam), Goat anti-CPA1 at 1:200 (R&D systems), guinea pig anti-Insm1 at 1:500 (provided by C. Birchmeier, Max-Delbrück-Centrum for Molecular Medicine, Berlin, Germany), rabbit anti-MafA at 1:2000 (Bethyl) and rabbit anti-MafB at 1:2000 (provided by R. Stein, Vanderbilt University, Nashville, TN, USA). Secondary antibodies conjugated to DyLyght-488, DyLight-549 and DyLight-649 (Jackson ImmunoResearch Laboratories) were used at 1:500. For mouse anti-Ki-67, signal amplification was performed using biotin anti-mouse coupled antibody at 1:200 (Vector Laboratories) and streptavidin-Cy3 conjugate at 1:500 (Molecular Probes). For antiβ-galactosidase immunofluorescence reactions were performed with tyramide signal amplification (TSA) (PerkinElmer). Nuclei were stained with DAPI and slides were mounted in Aqua-Poly/Mount (Polysciences). Immunohistochemistry was also performed on wax sections after whole mount X-gal staining and the primary antibodies were detected with the ABC immunoperoxidase/DAB system (Vector Lab. Inc., USA) Supplementary materials related to this article can be found online at doi:10.1016/j.ydbio.2011.10.025. Acknowledgments We are very grateful to Drs Guoquiang Gu and Douglas A. Melton for sharing the Neurog3 CreERT/+ mouse line. We thank members of the Gradwohl lab, Maria Elena Tores-Padilla, Guoquiang Gu and Jorge Ferrer for helpful discussions. We also thank Viviane Lobstein for excellent technical assistance. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), funding to G.G from the National Institutes of Health (NIH)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Beta cell Biology Consortium U19-DK061244 and U19-DK072495, the Juvenile Diabetes Research Foundation (JDRF) (AIP Cellules souches A03139MS), the Association Française des Diabétiques (AFD) and the European Union Integrated Project 6th FP Beta Cell Therapy LSHB-CT-2005-512145. A.B. has been a recipient of a Fellowship from the Fondation pour la Recherche Médicale. References Ahn, S., Joyner, A.L., 2004. Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118, 505–516. Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D.J., Honjo, T., Hrabe de Angelis, M., Lendahl, U., Edlund, H., 1999. Notch signalling controls pancreatic cell differentiation. Nature 400, 877–881. Cras-Meneur, C., Li, L., Kopan, R., Permutt, M.A., 2009. Presenilins, Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window. Genes Dev. 23, 2088–2101. D'Amour, K.A., Bang, A.G., Eliazer, S., Kelly, O.G., Agulnick, A.D., Smart, N.G., Moorman, M.A., Kroon, E., Carpenter, M.K., Baetge, E.E., 2006. Production of pancreatic hormoneexpressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392–1401. Desgraz, R., Herrera, P.L., 2009. Pancreatic neurogenin 3-expressing cells are unipotent islet precursors. Development 136, 3567–3574. Gouzi, M., Kim, Y.H., Katsumoto, K., Johansson, K., Grapin-Botton, A., 2011. Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development. Dev. Dyn. 240, 589–604. Gradwohl, G., Dierich, A., LeMeur, M., Guillemot, F., 2000. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. U. S. A. 97, 1607–1611. Gu, G., Dubauskaite, J., Melton, D.A., 2002. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447–2457. Hayashi, S., McMahon, A.P., 2002. Efficient recombination in diverse tissues by a tamoxifeninducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244, 305–318. Heller, R.S., Jenny, M., Collombat, P., Mansouri, A., Tomasetto, C., Madsen, O.D., Mellitzer, G., Gradwohl, G., Serup, P., 2005. Genetic determinants of pancreatic epsilon-cell development. Dev. Biol. 286, 217–224. Jacquemin, P., Durviaux, S.M., Jensen, J., Godfraind, C., Gradwohl, G., Guillemot, F., Madsen, O.D., Carmeliet, P., Dewerchin, M., Collen, D., Rousseau, G.G., Lemaigre, F.P., 2000.

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