Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells

Authors


Abstract

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

INTRODUCTION

Pancreatic islet cell transplantation is today an established procedure to restore the required mass of functional β-cells and long-term normoglycemia in Type 1 diabetic patients (Ryan et al., 2001; Shapiro et al., 2000), and this therapy could be an attractive option also for patients with severe type 2 diabetes. The use of this therapy is, however, restricted due to a shortage of transplantable material. One attractive alternative source for transplantable β-cells as a cure for diabetes involves the generation of functional β-cells from stem and/or progenitor cells. Apart from the identification and isolation of suitable stem or progenitor cells, being of embryonic, fetal, or adult origin, the factors that allow us to expand and ultimately induce the differentiation of these stem/progenitor cells into functional β-cells, must be identified. Hence, to realise the full potential of stem cells, the molecular mechanisms controlling these processes need to be elucidated. Throughout development, cell proliferation and differentiation have to be carefully regulated to ensure proper generation of functional organs and tissues. Such developmental processes are controlled, temporally and spatially, by means of various signalling pathways. The pancreas develops by means of evaginations of the primitive gut epithelium resulting in the formation of the dorsal and ventral pancreatic anlagen. The pancreatic progenitor cells present in these early pancreatic anlagen coexpress the transcription factors IPF1/PDX1, Nkx2.2, Nkx6.1, and p48 (Chiang and Melton, 2003), and subsequently proliferate and differentiate thus eventually giving rise to all differentiated, pancreatic epithelial cell types present in the mature pancreas (Edlund, 2002).

We have identified previously the Notch-signalling pathway as a mechanism through which pancreatic endocrine cell fate is regulated (Apelqvist et al., 1999). Studies on Hes-1 (Jensen et al., 2000), neurogenin (ngn) 3 (Gradwohl et al., 2000), Dll-1, and RBP-Jκ-deficient mice (Apelqvist et al., 1999), as well as mice overexpressing ngn3 in early pancreatic progenitors (Apelqvist et al., 1999; Schwitzgebel et al., 2000), collectively demonstrate that Notch signalling controls the choice between differentiated endocrine and progenitor cell fates in the developing pancreas. These data also reveal that ngn3 not only is competent to promote, but also required for, pancreatic endocrine cell differentiation. During pancreatic cell development, impaired Notch receptor activation or signalling results in profound ngn3 gene expression, leading to premature endocrine cell differentiation at the expense of pancreatic cell expansion and exocrine cell differentiation (Apelqvist et al., 1999; Jensen et al., 2000). In contrast, cells with active Notch-signalling most likely remain as undifferentiated progenitor cells that would allow the subsequent proliferation, morphogenesis, and later differentiation of pancreatic epithelial cells analogous to the function of Notch-signalling during early mammalian neurogenesis (Lewis, 1996; Beatus and Lendahl, 1998).

FGF-signalling plays a key role in the development of the mouse embryo and has been implicated in the development of many organs that are dependent on epithelial–mesenchymal interactions (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). The pancreas is an organ whose growth, branching morphogenesis, and differentiation are dependent on epithelial–mesenchymal interactions, thus implicating a potential role for FGFs and/or other growth factors (Edlund, 2002). We have shown previously that FGFR1c signalling in the adult mouse β-cells is required for normal β-cell function and maintenance of normoglycemia (Hart et al., 2000), and other studies have suggested a role for FGF-signalling during pancreatic development (Celli et al., 1998; Miralles et al., 1999; Ohuchi et al., 2000; Bhushan et al., 2001; Revest et al., 2001; Elghazi et al., 2002). Mice that express a dominant negative FGFR2b under the control of the Metallothionein promoter (Celli et al., 1998), and mice that lack FGFR2b (Revest et al., 2001) or Fgf10 (Ohuchi et al., 2000; Bhushan et al., 2001), a high-affinity ligand for FGFR2b, all show pancreatic hypoplasia. In vitro cultures of rat pancreatic anlagen have suggested that signalling by means of the FGFR2b stimulates exocrine differentiation and pancreatic endocrine progenitor cell proliferation (Miralles et al., 1999; Elghazi et al., 2002). We here show that persistent expression of the FGFR2b high-affinity ligand Fgf10 in the developing pancreatic epithelium of transgenic mice results in enhanced, prolonged proliferation of pancreatic epithelial cells, pancreatic hyperplasia, and impaired pancreatic cell differentiation. Moreover, our findings suggest that the effects exerted by Fgf10 perturb the lateral inhibition process and maintains Notch activation throughout the pancreatic epithelium.

RESULTS

Perturbed Pancreatic Development in Ipf1/Fgf10 Transgenic Mice

FGFR2 was expressed in nondifferentiated epithelial pancreatic cells between embryonic day (e) 9 and e13 but not in differentiated endocrine cells that appear at these stages (Fig. 1A–C). At later embryonic stages, the expression of FGFR2 becomes restricted such that by the neonatal stage, continuing through to the adult, the expression is limited to insulin-producing cells (Fig. 1D–F). The FGFR2b high-affinity ligand Fgf10 is highly expressed in the mesenchyme surrounding the pancreatic buds at early stages of development (Bhushan et al., 2001). We also observed a low-level Fgf10 expression within the pancreatic epithelium at later embryonic stages (Supplementary Fig. 1, which is available online at http://www.interscience.wiley.com/developmentaldynamics/suppmat/index.html). To begin to define the mechanism by which FGF10/FGFR2 signalling controls pancreatic progenitor cell proliferation and differentiation, we here used the Ipf1/Pdx1 promoter to generate transgenic mice expressing Fgf10 within the pancreatic epithelium throughout pancreatic development.

Figure 1.

Fibroblast growth factor receptor (FGFR) 2 is expressed in the developing pancreatic epithelium. A–F: Confocal analyses of the expression of FGFR2 at embryonic day (e) 9.5 (A), e11.5 (B), e13 (C), e15 (D), neonatal (E, neo), and adult (F) stages show that FGFR2 is expressed in pancreatic progenitor cells at early stages of development but then becomes gradually restricted to pancreatic β-cells. Glu, glucagon; Ins, insulin. C applies to Scale bars = 0.04 mm in C applies to A–D, 0.03 mm in F.

Transgene expression was confirmed by in situ hybridisation and real-time polymerase chain reaction (PCR; Supplementary Fig. 1 and data not shown) and these expression analyses also showed that the pancreatic expression of Fgf10 was increased two- to fourfold in transgenic mice compared with that of normal mice. Ipf1/Fgf10 transgenic mice were born alive and consistently smaller in size compared with their control littermates (data not shown). The transgenic pups appeared dehydrated, and the majority died at postnatal day (P) 1 or within the first postnatal week. Measurement of blood glucose levels of P1–P3 neonates revealed elevated blood glucose levels indicative of hyperglycemia in the transgenic pups (data not shown). Dissections of 15-day-old embryos and neonates showed a macroscopically malformed pancreas that appeared greatly increased in size compared with wild-type littermates (Fig. 2A–D). Apart from the overall, enlarged size of the pancreas, it also appeared more solid and condensed, unlike the characteristic loose and fluffy structure observed in pancreas dissected from wild-type littermates (Fig. 2A–D). These findings show that persistent, high-level expression of Fgf10 in the developing pancreas results in pancreatic hyperplasia.

Figure 2.

Pancreatic hyperplasia in Ipf1/Fgf10 mice. A–D: Gross morphologic analyses of embryonic day (e) 15 (A,B) and neonatal (C,D) wild-type (A,C), and Ipf1/Fgf10 transgenic (B,D) mice demonstrating the enlarged pancreas, more tightly packed appearance of the transgenic pancreas compared with that of stage-matched wild-type littermates. The size of the pancreata in A and B is indicated by the dotted lines. St, stomach; Sp, spleen; Dp, dorsal pancreas; Vp, ventral pancreas; d, duodenum.

Progressive Pancreatic Hyperplasia in Ipf1/Fgf10 Transgenic Mice

The increase in pancreatic cellular density in the transgenic mice compared with that of stage-matched wild-type was further demonstrated by histologic analyses involving the visualization of nuclei by DAPI, which confirmed the increased cellular density in the Ipf1/Fgf10 transgenic compared with stage-matched wild-type littermates (Fig. 3A–F). At e13, no drastic difference in the structure or cellular density of the transgenic compared with the wild-type pancreas was discernible (Fig. 3A,D). By e17, the Ipf1/Fgf10 transgenic pancreas appeared, however, not only increased in size compared with stage-matched littermates but also displayed a more dense pancreatic epithelial structure (Fig. 3B,E) and the difference both in size and epithelial structure was further pronounced at the neonatal stage (Fig. 3C,F). Hematoxylin-eosin (HE) staining of different stages of Ipf1/Fgf10 transgenic and wild-type pancreata confirmed the increasingly condensed structure of the pancreatic epithelium in the transgenic mice and also suggest that the transgenic epithelium is immature with poorly developed acinar structures (Fig. 3G–L and Supplementary Fig. 2, available online at http://www.interscience.wiley.com/developmentaldynamics/suppmat/index.html). The increase in overall size and the condensed structure of the transgenic pancreatic epithelia at stages later than e13 show that persistent, high-level FgfF10 expression results in progressive pancreatic hyperplasia.

Figure 3.

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

Increased Proliferation of Pancreatic Epithelial Cells in Ipf1/Fgf10 Mice

To determine the extent of epithelial cell proliferation in the Ipf1/Fgf10 mice compared with that of controls, we next performed double-immmunohistochemical analyses by using antibodies against the ductal-specific marker cytokeratin-7 and antibodies specific for the mitotic marker phospho-Histone H3 on e17 pancreata (Fig. 4A–F). In the Ipf1/Fgf10 mice, cytokeratin-7 was expressed throughout the epithelium in contrast to the more restricted, patchy expression observed in the wild-type pancreas (Fig. 4A,D and Supplementary Fig. 3, available online at http://www.interscience.wiley. com/developmentaldynamics/ suppmat/index.html). Mitotically active phospho-H3–positive cells were found uniformly throughout the e17 Ipf1/Fgf10 pancreatic epithelium, whereas only a few scattered phospho-H3–positive cells were detected in the pancreata of control mice. The number of phospho-Histone H3–positive cells/pancreatic area was increased by 50% e17 Ipf1/Fgf10 mice compared with stage-matched controls, and the increase in the total pancreatic area in the transgenic mice at this stage was ∼30% compared with controls. Together these data provide evidence that the pancreatic hyperplasia observed in the Ipf1/Fgf10 transgenic mice results from an enhanced proliferation of the pancreatic epithelial cells.

Figure 4.

Enhanced proliferation of pancreatic epithelial cells in Ipf1/Fgf10 mice. A–F: Cytokeratin-7 (A,C,D,F) and phospho Histone H3 (PH3, B,C,E,F) immunostaining of embryonic day (e) 17 wild-type (A–C) and Ipf1/Fgf10 transgenic (D–F) pancreata reveal a perturbed organization of the transgenic epithelium and an increased pancreatic epithelial cell proliferation in Ipf1/Fgf10 compared with wild-type mice. Scale bar = 0.05 mm in A (applies to A–F).

Impaired Differentiation of Pancreatic Cell Types in Ipf1/Fgf10 Mice

To analyze in detail the differentiated state of the transgenic pancreas at the neonatal stage, we next examined the expression of transcription factors, hormones, and enzymes. Immunohistochemical analyses of neonatal pancreas by using antibodies specific for insulin (Ins), glucagon (Glu), and somatostatin (data not shown) confirmed that pancreatic endocrine cell differentiation was severely perturbed in the transgenic mice (Fig. 5A,B). In normal mice, endocrine cells cluster into distinct islets (Fig. 5A). The Ipf1/Fgf10 transgenic mice displayed drastically fewer (less than 1% of that of wild-type pancreas) hormone-producing cells that failed to cluster (Fig. 5B). Consequently, the expression of the transcription factor ISL1, a marker for differentiated pancreatic endocrine cells (Edlund, 2002), was mainly expressed in few, nonclustered cells corresponding to the few hormone-producing cells that formed in the transgenic pancreata, as opposed to the expression detected in the clustered pancreatic endocrine cells of control pancreata (Fig. 5C,D). Together these data provide evidence that pancreatic endocrine cell differentiation is perturbed in Ipf1/Fgf10 transgenic mice. The expression of exocrine enzymes such as carboxypeptidase A (Fig. 5E,F) was reduced in Ipf1/Fgf10 mice compared with that observed in control pancreata. The condensed, abnormal organization of the pancreatic epithelia, perturbed pancreatic acinar formation, uniform cytokeratin-7 expression, and diminished exocrine marker expression in Ipf1/Fgf10 mice compared with control mice (Figs. 3, 5E,F) suggest that differentiation of pancreatic epithelial cells into exocrine cells also is perturbed in Ipf1/Fgf10 mice. Taken together, the increased pancreatic epithelial cell proliferation and impaired pancreatic cell differentiation observed in Ipf1/Fgf10 mice raise the possibility that the majority of these pancreatic epithelial cells may represent pancreatic progenitors.

Figure 5.

Impaired pancreatic cell differentiation in Ipf1/Fgf10 mice. A–L: Immunohistochemical analyses of differentiated pancreatic markers in wild-type (A,C,E,G, I,K,) and Ipf1/Fgf10 transgenic (B,D,F,H,J,L) neonates show in contrast to control mice only occasional cells expressing pancreatic endocrine markers such as glucagon (Glu, A,B), insulin (Ins, A,B,I,J), and ISL1 (C,D) were detected in the transgenic pancreas. Carboxypeptidase A (CPA, E,F) expression was perturbed in the transgenic pancreas, which also lacked distinct acinar structures. A low but uniform expression of IPF1 (G,H), and Nkx6.1 (I,J) appeared throughout the transgenic pancreas compared with the strong and β-cell–specific expression seen in the wild-type pancreas. The transgenic pancreatic epithelium also expressed Ptf1a/p48 (p48, K,L). Scale bars = 0.04 mm in A (applies to A–D,G–L), 0.05 mm in E (applies to E,F).

Ipf1/Fgf10 Pancreatic Epithelial Cells Express Pancreatic Progenitor Cell Markers

Single-cell transcriptional profiling of early pancreatic epithelial cells (Chiang and Melton, 2003) together with the expression profile of different pancreatic transcription factors (Edlund, 2002) suggests that early pancreatic stem and/or progenitor cells coexpress the transcription factors IPF1/PDX1, Nkx6.1, Nkx2.2, and p48. The transcription factor IPF1/PDX-1 is normally highly expressed in early pancreatic progenitor cells, at lower levels in epithelial cells during the phase of massive pancreatic epithelial cell expansion (i.e., from ∼e10 and onward), and then becomes highly expressed in mature β-cells with barely detectable expression in other differentiated pancreatic cell types (Ohlsson et al., 1993; Edlund, 1998). Ipf1/Fgf10 neonates displayed a uniform pancreatic epithelial expression of IPF1/PDX1, reminiscent of the lower level expression of IPF1/PDX1 in proliferating epithelial cells (Ohlsson et al., 1993; Edlund, 1998) with only occasional high-level IPF1/PDX1-expressing cells (Fig. 5H). In control mice, IPF1/PDX1 was highly expressed in the β-cells with only a low, barely detectable expression in other pancreatic cell types (Fig. 5G). The Nkx-transcription factors Nkx6.1 (Fig. 5I,J) and Nkx2.2 (not shown), which are expressed in the early pancreatic progenitor cells and later become restricted to β-cells (Nkx6.1), or all endocrine cells with the exception of δ-cells (Nkx2.2; Sussel et al., 1998; Sander et al., 2000), were also expressed at a low level in Ipf1/Fgf10 pancreatic epithelial cells. Occasional cells expressing high levels of these transcription factors, representing the few differentiated endocrine cells that still appear in the Ipf1/Fgf10 mice, could also be observed in the transgenic pancreas (data not shown). The pancreatic transcription factor Ptf1a/p48 (Krapp et al., 1998), which is expressed in early pancreatic progenitor cells from ∼e10 (Selander and Edlund, 2002; Kawaguchi et al., 2002) and later becomes restricted to differentiated exocrine cells (Krapp et al., 1998), was also expressed throughout the pancreatic epithelium of transgenic neonates (Fig. 5K,L). The expression of IPF1/PDX1, Nkx6.1, Nkx2.2, and P48 in Ipf1/Fgf10 pancreatic epithelial cells is supportive of the immature, progenitor-like nature of these cells.

Expression of Notch-Signalling Components Is Perturbed in Ipf1/Fgf10 Mice

To investigate whether the impaired cell differentiation in the Ipf1/Fgf10 mice reflected a perturbation in the induction of pancreatic cell types or merely a block in their terminal differentiation, we next analyzed the expression of Notch signalling components in the transgenic pancreata. During pancreatic development, ngn3 expression marks endocrine progenitors and its expression is regulated by means of the Notch-signalling pathway (Apelqvist et al., 1999; Jensen et al., 2000). Analyses revealed that ngn3 expression was impaired in Ipf1/Fgf10 mice not only at neonatal stages (data not shown) but also at e15 (Fig. 6A,B) compared with stage-matched littermates, suggesting that pancreatic endocrine cell differentiation is impaired already at the endocrine progenitor stage. Consistent with the reduced number of ngn3 expressing cells, Dll1 expression also appeared reduced compared with wild-type pancreas (Fig. 6C,D). No increased cell apoptosis was observed as determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay or by staining for the apoptotic marker Caspase 3 (data not shown), suggesting that the decreased number of pro-endocrine and endocrine cells in Ipf1/Fgf10 mice is not caused by cell death but rather by impaired specification of pro-endocrine cells. In the wild-type pancreas, Notch1 was highly expressed predominantly in the developing acini at e15 (Fig. 6E,F) but absent from the streaks of differentiating endocrine cells (data not shown and Apelqvist et al., 1999). In the Ipf1/Fgf10 mice, Notch1 expression appeared to be expressed throughout the pancreatic epithelium, reminiscent of the expression of Notch1 in e13 pancreatic epithelium (Apelqvist et al., 1999). The uniform expression of Notch1 in the Ipf1/Fgf10 pancreatic epithelia was matched by the expression of HES-1 (Fig. 6G,H), suggesting that Notch activation is maintained throughout the pancreatic epithelium of Ipf1/Fgf10 mice. A uniform, maintained activation of Notch activation is consistent with the impaired pancreatic expression of ngn3 in Ipf1/Fgf10 mice. Together these data show that persistent expression of Fgf10 impairs the expression of the pancreatic pro-endocrine gene ngn3, which in turn suggests that the Notch-mediated lateral inhibition pathway is impaired in the Ipf1/Fgf10 mice. The lack of differentiated cell types in the Ipf1/Fgf10 mice might reflect a deregulation of Notch-mediated lateral inhibition pathway where sustained Notch activation would “lock” a majority of the pancreatic epithelial cells in a nondifferentiated, progenitor-like state with maintained proliferative capacity.

Figure 6.

The expression of the pro-endocrine marker ngn3 expression is drastically perturbed in Ipf1/Fgf10 transgenic mice. A–J: In situ hybridisation analyses of embryonic day (e) 15 wild-type (A,C,E,G,I) and Ipf1/Fgf10 transgenic (B,D,F,H,J) pancreata show that ngn3 (A,B) expression is severely impaired in the transgenic mice. Dll1 (C,D) expression in Ipf1/Fgf10 transgenic mice is also reduced compared with that of stage-matched wild-type pancreas, whereas Notch1 (E,F) and HES-1 (G,H) is expressed throughout the ductal epithelium of the transgenic pancreas. Sel-1l (I,J) is strongly expressed in the forming acinar structures of e15 wild-type pancreas, whereas Sel-1l expression is reduced in the pancreatic epithelium of Ipf1/Fgf10 transgenic mice. Scale bar = 0.05 mm in A–J.

Genes that regulate Notch activity include sel-1, which in C. elegans has been shown to act as a negative regulator of Notch activity (Grant and Greenwald, 1997). Sel-1l, the mouse homologue (Donoviel et al., 1998) of C. elegans sel-1, starts to be expressed in the developing pancreatic acini from ∼e13, and Sel-1l remains highly expressed in exocrine cells at later stages of development and in the adult pancreas (Fig. 6I and data not shown; Donoviel et al., 1998), implicating a role for Sel-1l during pancreatic cell differentiation. In contrast to the high level of pancreatic expression of Sel-1l in the developing acini of e15 control mice, Sel-1l was expressed at an apparently lower level in the pancreas of e15 Ipf1/Fgf10 transgenic mice (Fig. 6J). Together these findings suggest that persistent ductal epithelial expression of Fgf10 perturbs the expression of the Notch antagonist Sel-1l.

DISCUSSION

Here, we show that persistent expression of the FGFR2b high-affinity ligand Fgf10 in the developing pancreatic epithelium of transgenic mice leads to pancreatic hyperplasia and that the pancreatic epithelium of these mice is largely immature. The gain-of-function data presented here and the previously reported pancreatic hypoplasia of mice lacking Fgf10 (Ohuchi et al., 2000; Bhushan et al., 2001) or perturbed FGFR2b function (Celli et al., 1998; Revest et al., 2001) together strongly support a key role for FGF10/FGFR2 signalling in stimulating pancreatic epithelial cell expansion.

Our data provide evidence that Fgf10 stimulates enhanced, prolonged proliferation of pancreatic epithelial cells, and impairs pancreatic cell differentiation. Moreover, our findings suggest that one of the effects exerted by Fgf10 involves a perturbation of the lateral inhibition process such that Notch activation is maintained throughout the pancreatic epithelium. The impaired expression of the pro-endocrine gene ngn3 is consistent with a maintained Notch activation and provides an explanation for the perturbed differentiation of pancreatic endocrine cells observed in the transgenic mice. The condensed, atypical organization of the pancreatic epithelia in Ipf1/Fgf10 mice together with the uniform expression of cytokeratin-7 and impaired expression of exocrine enzymes show that pancreatic exocrine differentiation also is perturbed in these mice. In addition to cytokeratin-7, the majority of the cells in the pancreatic epithelium of the Ipf1/Fgf10 mice express Ptf1a/p48, low levels of IPF1/PDX1, Nkx6.1 and Nkx2.2, i.e., transcription factors that are expressed in pancreatic progenitor cells (Chiang and Melton, 2003). These cells do not express endocrine markers like Isl1 and, although IPF1/PDX1 and Nkx6.1 are expressed also in differentiated β-cells, Ptf1a/p48 is not. Taken together, the impaired pancreatic cell differentiation, pancreatic ductal hyperplasia, sustained pancreatic cell proliferation, and the expression of pancreatic progenitor cell markers in the transgenic pancreatic epithelium, suggest that the proliferating pancreatic epithelial cells of the Ipf1/Fgf10 mice may possess progenitor-like properties.

The observation that Notch activation appears to be maintained in the pancreatic epithelium of Ipf1/Fgf10 mice suggests that the effect of FGF10/FGFR2b signalling on pancreatic epithelial cell proliferation and differentiation involves the Notch signalling pathway. Cells with activated Notch would be inhibited to differentiate and instead maintain the ability to proliferate, resulting in a sustained pancreatic epithelial cell proliferation throughout pancreatic development. The role for Fgf10 during pancreatic development might thus be dual; to maintain the pancreatic progenitor cells in an undifferentiated state and to provide the proliferative cue. During normal pancreatic development Fgf10 is predominantly expressed in the mesenchyme and as pancreatic development progresses the mesenchyme/epithelium ratio decreases. Hence, at later stages of pancreatic development the concentration of Fgf10 ligand may no longer be sufficient to block differentiation and stimulate proliferation and as a consequence cellular differentiation increases. It should also be stressed that, although Fgf10 appears to be crucial for pancreatic growth (Ohuchi et al., 2000; Bhushan et al., 2001), other factors, including other FGFs and the EGF-family of signalling factors, have also been suggested in pancreatic cell proliferation (reviewed in Edlund, 2002), indicating that FGF10 may act in concert with other factors to effectively stimulate pancreatic progenitor cell proliferation.

The reduced expression of Dll1 and ngn3, together with the maintained expression of Notch1 and HES-1 in the pancreatic epithelium of Ipf1/Fgf10 mice, suggests that the stimulatory effect of Fgf10 on pancreatic epithelial cell expansion, directly or indirectly, involves Notch signalling. Studies of neuronal differentiation in vitro are supportive of a mechanism where FGFs stimulate proliferation and inhibit differentiation by means of the Notch pathway (Faux et al., 2001). The differentiation of neuroepithelial precursor cells can be impaired by addition of either soluble Delta1 or FGFs (Faux et al., 2001). The addition of FGF1 or 2 to neuroepithelial precursor cells resulted in an up-regulation of Notch expression and a decrease in Delta1 expression (Faux et al., 2001). Our data suggest that not only is the Notch-mediated lateral inhibition mechanism conserved between the neuronal system and the pancreas (Lewis, 1996; Beatus and Lendahl, 1998; Apelqvist et al., 1999; Grandwohl et al., 2000; Jensen et al., 2000) but also the potential role for FGFs as moderators of Notch-signalling.

Genetic evidence from studies of C. elegans sel-1 suggests that sel-1 is a negative regulator of Notch (Grant and Greenwald, 1997). Based on its homology to a gene in yeast, HRD3, which is required for degradation of the HMG-CoA reductase, sel-1 has been proposed to regulate turnover of Notch, suggesting that absence of sel-1 may result in an atypical accumulation of activated Notch receptor (Grant and Greenwald, 1997). In the mouse pancreas, Sel-1l expression starts to be detectable from e13 and is highly expressed in the developing pancreatic epithelium at this and later stages of pancreatic development and in the adult pancreas (Donoviel et al., 1998, Fig. 6I and data not shown). In the Ipf1/Fgf10 mice, Sel-1l expression appears reduced, raising the possibility that continued high expression of Fgf10 perturbs the initiation and/or level of Sel-1l expression. If the role for Sel-1l in the developing pancreas is to antagonise activated Notch, thus allowing cell differentiation, then reduced Sel-1l expression may result in an atypical maintenance of activated Notch that would impair cell differentiation. The exact role for Sel-1l in relation to Notch activity and pancreas development need, however, to be elucidated through genetic manipulation of Sel-1l function in mice.

EXPERIMENTAL PROCEDURES

Generation of Ipf1/FGF10 Mice

A 4.5-kilobase (kb) NotI-NaeI fragment located immediately upstream of the Ipf1/Pdx1 gene (Apelqvist et al., 1997) was subcloned into a vector carrying a polyA site and a 750-base pair (bp) NotI/NcoI fragment that corresponded to the full-length mouse FGF10 cDNA. Thus, spatial and temporal expression of the ligand throughout development was ensured. We generated transgenic mice by pronuclear injection of the purified (NotI/BamHI; 1.8 ng/ml) into F2 b6/CBA hybrid oocytes as described (Hogan et al., 1994). Eight Ipf1/Fgf10 founders were obtained from 2 days of injections. Of the eight initial founders, one died at the neonatal stage, presenting the phenotype described in the study. From the resulting seven transgenic founder mice, two failed to transmit the transgene, whereas the remaining five (three males, two females), when bred with wild-type mice, transmitted the transgene to offspring that presented with the phenotypes described in the study.

Genotyping

The genotype of all offspring was determined by PCR analysis of genomic DNA extracted from tail biopsies or the yolk sac by proteinase K (Boehringer) digestion and isopropanol precipitation. The 5′ and 3′ primers used (amplified ∼500 bp) were 5′-TAGCGAGGGGGAAGAGGAGAT-3′ (Ipf1/Pdx-1 primer for 5′) and 5′-CTTACAGCTCCCAAGGGAATC-3′ (the FGF10 primer for 3′). The PCR conditions used were as follows: 1 cycle of 96°C for 5 min, 55°C for 2 min, and 72°C for 3 min, followed by 29 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 3 min; and finally 1 cycle of 72°C for 10 min.

In Situ Hybridization and Immunohistochemistry

In situ hybridization by using DIG-labelled RNA probes for ngn3 (Apelqvist et al., 1997), Notch1 (Apelqvist et al., 1999), Dll1 (Apelqvist et al., 1999), HES-1 (Apelqvist et al., 1999), Sel-1L (Donoviel et al., 1998), and FGF10 (Bellusci et al., 1997) was performed essentially as described (Ahlgren et al., 1996). Fgf10 in situ pictures in gray mode were treated in false color by using the single channel LUT display of a NIKON EZ-C1 program (1.6 build 87, C1 hardware version 1.0, Nikon Corporation © 2002), and the incorporate scale was used to estimate the expression levels. Immunohistochemical localization of antigens was performed as described (Ahlgren et al., 1996; Hart et al., 2000). The primary antibodies used were rabbit anti-IPF1 (Ohlsson et al., 1993), rabbit anti-ISL1 (Thor et al., 1991), rabbit anti-FGFR2 (Research Diagnostics, Inc.), rabbit anti-phospho Histone H3 (Upstate Biotechnology), rabbit anti-carboxypeptidase A (Anawa), rabbit Nkx6.1 (raised against a GST-Nkx6.1 fusion protein by AgriSera AB), rabbit anti-Nkx2.2 (Briscoe et al., 1999), rabbit anti-p48 (Li and Edlund, 2001), mouse monoclonal anti-cytokeratin 7 (DAKO), guinea pig anti-insulin (Linco), guinea pig anti-glucagon (Linco), rabbit anti-glucagon (Euro-Diagnostica), and rabbit anti-cleaved caspase 3 (Cell Signalling). The secondary antibodies used were fluorescein anti-guinea pig (Jackson), fluorescein anti-mouse (Jackson), and Cy3 anti-rabbit (Jackson). Quantification of total pancreatic area was performed on three independent transgenic e17 pancreata by using a Zeiss Axionplan 2 imaging microscope at a magnification of ×5. The proliferating cell cross-sectional area and total pancreatic area were measured by using the Image-Pro Plus 4.1 image analysis software (Media Cybernetics). At least 40 sections, separated by 500 μm, were measured per animal.

Confocal Microscopy

Longitudinal sections of dorsal pancreata were immunostained as described above. Images were collected on a Leica TCS SP confocal microscope fitted with spectrophotometer for emission band wavelength selection and dual detectors and argon/krypton (Ar/Kr) and Gre/Ne lasers for simultaneous scanning of two different fluorochromes. Confocal microscopy was used to detect the FGFR2 expression pattern in wild-type. Sets of fluorescent images were acquired simultaneously for Cy3- and fluorescein-tagged anti-FGFR2 and anti-insulin or anti-glucagon markers, respectively. Companion images were scanned with pixel size 1 and 0.7-μm step size. Confocal image stacks were combined as x–y projection images, digitally optimized, and assigned red and green pseudocolours for Cy3 and fluorescein, respectively.

Glucose Measurements

Blood samples were obtained from the tail vein from nonfasted animals (wild-type and transgenic) and glucose levels were measured by using a Precision Plus glucometer (Medisense).

RNA Extraction and Reverse Transcription

Total RNA was extracted from approximately 10–20 mg of pancreatic tissues from e15 embryos after homogenisation by using the NucleoSpin RNA II kit (BD Biosciences) following the manufacturer's instructions. RNA was reverse-transcribed by using the SuperScript First-Strand Synthesis kit (Invitrogen) again following the manufacturer's instructions.

Real Time PCR

Expression levels of fgf10 mRNA in transgenic (n = 3) and wild-type mice (n = 4) were determined by using the ABI PRISM 7000 Sequence Detection System. PCR reactions were carried out in 25-μl volumes in 96-well plates, in a reaction buffer containing 1× SYBR Green PCR Master Mix and 300 nmol of primers. All reactions were run in duplicates with a preoptimised control primer pair for β-actin, which enabled data to be expressed in relation to an internal reference, to allow for differences in RT efficiency. Primers used for β-actin were 5′-GGCCAACCGTGAAAAGATGA-3′ and 5′-ACGTAGCCATCCAGGCTGTG-3′, for Fgf10 were 5′-CAGCGGGACCAAGAATGAAG-3′ and 5′-TGACGGCAACAACTCCGATTT-3′. All primers annealed at 59°C and yielded amplicons of 70–150 bp. Reaction conditions were as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 sec, and 60°C for 1 min. We quantified expression levels by using the comparative Ct method. According to the manufacturer's guidelines, data were expressed as Ct values (the cycle number where logarithmic PCR plots cross a calculated threshold line) and used to determine ΔCt (ΔCt = Ct of the target gene, e.g., Ct of Fgf10 minus the Ct of the housekeeping gene). Fold difference in expression levels with relation to the reference gene was calculated by using the equation 2-Δ ΔCt (ΔΔCt = ΔCt Sample − ΔCt calibrator). Measurements were carried out a minimum of three times each and showed a 1.5- to 2-fold increase in Fgf10 expression in the transgenic compared with wild-type mice.

Acknowledgements

We thank Dr. Dorit Donoviel for the Sel-1l cDNA, Dr. Thomas Edlund for critical reading and comments, and Dr. Stefan Norlin and other and members in our laboratory for helpful discussions. H.E. received funding from the Swedish Research Council; the Juvenile Diabetes Foundation, New York; the Wallenberg Consortium North; and the EU 5th program and EU regional fund, objective 1.

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