Exocrine-to-endocrine differentiation is detectable only prior to birth in the uninjured mouse pancreas
© Kopinke and Murtaugh; licensee BioMed Central Ltd. 2010
Received: 28 January 2010
Accepted: 8 April 2010
Published: 8 April 2010
Histological evidence suggests that insulin-producing beta (β)-cells arise in utero from duct-like structures of the fetal exocrine pancreas, and genetic lineage tracing studies indicate that they are maintained in the adult by self-renewal. These studies have not addressed the origin of the new β-cells that arise in large numbers shortly after birth, and contradictory lineage tracing results have been published regarding the differentiation potential of duct cells in this period. We established an independent approach to address this question directly.
We generated mice in which duct and acinar cells, comprising the exocrine pancreas, can be genetically marked by virtue of their expressing the mucin gene Muc1. Using these mice, we performed time-specific lineage tracing to determine if these cells undergo endocrine transdifferentiation in vivo. We find that Muc1+ cells do give rise to β-cells and other islet cells in utero, providing formal proof that mature islets arise from embryonic duct structures. From birth onwards, Muc1 lineage-labeled cells are confined to the exocrine compartment, with no detectable contribution to islet cells.
These results argue against a significant contribution by exocrine transdifferentiation to the normal postnatal expansion and maintenance of β-cell mass. Exocrine transdifferentiation has been proposed to occur during injury and regeneration, and our experimental model is suited to test this hypothesis in vivo.
The origin of pancreatic islet cells has been the subject of study and controversy since before the discovery of insulin [1–3]. Histological and ultrastructural studies suggested that islets arise from exocrine ducts during embryogenesis, but whether such exocrine-endocrine conversion continued after birth remained a matter of controversy . Nucleotide analogues have recently been used to identify and trace the fate of proliferating cells, but these studies have been interpreted both for and against the hypothesis of new islet cell differentiation, or neogenesis, in adulthood [5, 6]. Similarly, cell culture studies have provided evidence for and against neogenesis, but extrapolating these findings in vivo remains a challenge . Nonetheless, determining whether β-cell neogenesis occurs in vivo will inform efforts to replenish β-cells lost in diabetes: if neogenesis can occur in mice, it might be possible in the human organ as well. Evidence against neogenesis would encourage more aggressive efforts elsewhere, such as the derivation of β-cells from human embryonic stem cells.
Genetic lineage tracing techniques have transformed our understanding of pancreas developmental biology, providing insights either unavailable from or contradictory to prior studies of fixed tissue . For example, using the Cre-loxP system to monitor the fate of cells expressing the acinar protein Carboxypeptidase A1 (Cpa1) has revealed that these cells comprise a self-renewing, multipotent progenitor pool during mid-embryogenesis . At later stages, Cpa1+ cells become restricted to the acinar compartment, and lineage tracing of adult acinar cells indicates that they do not contribute to β-cells [7, 8]. Other lineage tracing studies similarly cast doubt on the neogenesis model; for example, it is now appreciated that all islet cell types arise from Neurog3+ precursor cells , yet Neurog3+ cells are usually not detected after birth [10–12]. Similarly, a key study of adult β-cell expansion and self-renewal suggests that these processes reflect division of existing β-cells rather than neogenesis , a conclusion reinforced by subsequent independent findings [5, 14].
Nonetheless, without a Cre line capable of marking duct cells, these studies could not exclude a minor ductal contribution to β-cells. Furthermore, they have not addressed the rapid expansion of β-cell mass that occurs shortly after birth , which may derive in part from neogenesis . The ideal tool to address these questions would be a mouse line in which ducts can be inducibly marked at a specific time point, and their ability to contribute to β-cells determined at later stages: a cellular "pulse-chase" experiment . Such mice could also clarify the origin of new β-cells formed in regeneration models, such as duct ligation and partial pancreatectomy, which may involve neogenesis [16, 17]
Four transgenic mouse lines have recently been described in studies aimed at addressing this issue. Inada et al.  generated mice in which either Cre or its tamoxifen (TM)-inducible derivative CreER™ is driven by the promoter of Carbonic anhydrase II (CAII), a marker of adult duct cells. This study concluded that ducts continue to give rise to islets after birth, and that neogenesis is dramatically increased after duct ligation . Means et al.  used a knock-in approach to target a similar tamoxifen-inducible CreERT molecule to the duct-specific cytokeratin-19 (K19) gene. K19 CreERT labels many duct cells in neonatal and adult mice, as well as a small fraction of islet cells due to "leaky" recombinase expression in islets themselves. When K19 CreERT was activated by TM treatment at birth, islet labeling at one week of age was no greater than expected from this background activity, suggesting that new islet cells generated in the interim did not derive from the more robustly-labeled duct population . Finally, Solar et al.  generated a BAC transgenic in which TM-dependent CreERT2 is targeted to the first exon of Hnf1β, a transcription factor expressed selectively in embryonic and adult duct cells. This study indicated that Hnf1β+ duct cells give rise to β-cells prior to birth but not thereafter, even in the context of injury models such as duct ligation . The apparent contradiction between these studies, in particular those using CAII- and Hnf1β-driven Cre transgenes, suggests that additional markers should be sought out and exploited for their capacity to label duct cells, thereby providing independent evidence for or against postnatal neogenesis .
The gene mucin1, transmembrane (Muc1) is expressed throughout the ductal network in both the embryonic and adult pancreas, and excluded from islets [22, 23]. We have generated mice in which the Muc1 gene is tagged with an IRES-CreERT2 cassette, permitting the inducible labeling of Muc1+ cells. We find that this line also marks embryonic and postnatal acinar cells, reflecting endogenous Muc1 expression, but is completely excluded from islets in short-term "pulse" experiments. In the embryo, Muc1+ cells give rise to endocrine cells as well as their Neurog3+precursors, formally confirming that islets originate from fetal ducts. Following birth, however, we find that the Muc1+ lineage completely fails to contribute to new islet α- or β-cells, indicating that both the expansion and homeostasis of these cell types occurs independent of contribution from exocrine ducts or acini. These results argue against a major role for neogenesis in the normal postnatal pancreas, and set the groundwork for studies of potential neogenesis during regeneration.
Targeting CreERT2 to the Muc1locus
As detailed in the Methods, we generated a Muc1IRES-CreERT2 allele (henceforth, Muc1IC2) by gene targeting, introducing an IRES-CreERT2 cassette after the endogenous stop codon of the Muc1 locus (Figure 3D-E). The CreERT2 protein is a tamoxifen (TM)-dependent recombinase that provides temporal control of Cre activity , which should allow us to investigate the differentiation potential of embryonic and postnatal duct cells. Muc1IC2 heterozygous and homozygous mice are viable and fertile (data not shown).
Muc1 IC2 marks exocrine cells in the adult pancreas
Examining TM-untreated 4-8 month Muc1IC2/+;Rosa26EYFP/+ and Muc1IC2/+;Rosa26LacZ/+ mice (n = 3) revealed a background recombination rate of less than 0.1% (Figure 4F and data not shown), confirming that this line exhibits stringent tamoxifen dependence. Furthermore, we never observed lineage-labeled endocrine cells in our short-term experiments. Muc1IC2 labeling is thus TM-dependent, as expected, and extends throughout the exocrine pancreas (acini and ducts). Although TM-induced Muc1IC2 labeling is relatively sparse (5-15% of exocrine cells, in this experiment), its distribution closely matches that of Muc1 itself, and appears to represent a random sampling of the exocrine pancreas. As Muc1IC2 does not directly label islet cells, it can be used to detect potential exocrine-derived neogenesis.
Islet cells arise from embryonic Muc1+cells
Altogether, these results strongly suggest that (a) embryonic Muc1+ duct cells give rise to all segments of the adult ductal network, (b) Muc1 is not expressed by endocrine cells and (c) mature islet cells arise from Neurog3+ precursors within the embryonic Muc1+ ductal network. These results represent direct evidence that endocrine cells arise from embryonic ducts, but they leave open the possibility that embryonic Muc1IC2 labeling actually marks a ductal stem cell-like population, which continues to give rise to new islet cells after birth. We therefore turned our attention to the differentiation potential of postnatal Muc1+ cells.
Muc1+exocrine cells do not undergo endocrine differentiation after birth
Muc1IC2 lineage contribution to exocrine cells
Lineage+(EYFP or LacZ)
% of DAPI+
% of AMY+
% of CK19+
Muc1 IC2/+ ; Rosa26 EYFP/+
10 mg TM
23.3 ± 0.7
29.6 ± 1.4
6.4 ± 0.6
24.0 ± 3.6
31.6 ± 5.3
11.6 ± 1.1
23.7 ± 2.2
30.8 ± 3.2
9.6 ± 1.2
Muc1 IC2/+ ; Rosa26 LacZ/+
10 mg TM
8.6 ± 2.4
11.2 ± 3.5
3.7 ± 1.6
17.7 ± 3.7
26.4 ± 4.5
5.8 ± 0.4
10.6 ± 2.5
15.5 ± 3.8
4.3 ± 1.2
Muc1 IC2/+ ; Rosa26 EYFP/+
2 mg TM
31.9 ± 9.6
10.1 ± 1.7
Quantification of potential Muc1 contribution to endocrine cells
number of cells scored
Muc1 IC2/+ ; Rosa26 EYFP/+
10 mg TM
Muc1 IC2/+ ; Rosa26 LacZ/+
10 mg TM
Muc1 IC2/+ ; Rosa26 EYFP/+
2 mg TM
To determine if Muc1+ cells contribute to the rapid expansion of islet cell numbers after birth , we performed experiment 2, in which we induced recombination in Muc1IC2/+; Rosa26LacZ/+ neonates by administration of tamoxifen to nursing mothers (consecutive 10 mg doses on P0 and P1). We sacrificed mice 21 or 120 days after treatment (i.e. as weanlings or mature adults), and performed immunofluorescence to detect LacZ lineage marker within the exocrine and endocrine compartments (Figure 10D-F and data not shown). We found an overall labeling efficiency of ~10% in these experiments, including ~15% of acinar cells and ~4% of duct cells (Table 1). As in experiment 1, however, despite scoring several thousand cells for each marker, we did not observe a single LacZ+ β-cell or α-cell (Table 2).
Previous studies indicate that acinar cells do not contribute to islets after birth [7, 8, 31], and experiment 2 suggests that neonatal duct cells are also excluded from the islet lineage. This interpretation hinges on relatively infrequent ductal labeling, which could have hidden a low level of duct-derived neogenesis. To increase the duct labeling frequency, we performed experiment 3, in which we directly injected newborn Muc1IC2/+; Rosa26EYFP/+ pups with tamoxifen (2 mg per pup, delivered subcutaneously). Upon sacrifice, 21 days after TM administration, we found increased overall labeling compared to mice that received maternal TM (~30% EYFP+, Table 1). Importantly, the duct labeling frequency was increased to 10%, with equal distribution among interlobular, intralobular and intercalated ducts (Figure 6D). Nonetheless, we did not observe any labeled β-cells or α-cells, despite scoring several thousand cells positive for each marker (Table 2).
These analyses suggest an upper limit to the contribution of neogenesis to postnatal islet growth. β-cell mass has been reported to expand between 4- and 10-fold in the first 2-4 weeks after birth [32–34]. If we assume a five-fold expansion between P0 and P21, we can infer that ~80% of the β-cells scored in experiment 3 were "new" since P0 (3600 of the ~4500 β-cells counted, Table 2). If all of these had been derived from Muc1IC2-labeled duct cells, given a duct labeling index of ~10% (Table 1), we would have expected to observe roughly 360 labeled β-cells. As we observed zero, we conclude that ≤1% of all β-cells generated after birth could have arisen from labeled ducts (1% neogenesis would have resulted in ~4 labeled β-cells, which is probably near the limit of reliable detection). Altogether, experiments 1-3 fail to reveal duct-to-islet transdifferentiation after birth.
We had intended, in creating the Muc1IC2 allele, to specifically address the differentiation potential of duct cells. Instead, we find that Muc1IC2 labels both acinar and duct cells, at all stages examined, and that Muc1 protein is readily detected within acinar cells. Nonetheless, we can treat the labeling of postnatal acinar cells as "background," as acinar-to-islet transdifferentiation does not occur after birth [7, 8, 31]. Cells expressing the acinar enzyme Cpa1 do behave as multipotent "tip cell" progenitors prior to E13.5, but are thereafter restricted to the acinar lineage . As Muc1+ cells still contribute to islets at E13.5 and E15.5 (Figs. 7, 9), we propose that islet differentiation competence normally shifts from Muc1+/Cpa1+ tips to Muc1+/Cpa1-negative "trunks" after E13.5, before being lost entirely at birth (Figure 11).
Another recently developed mouse line, K19 CreERT , in which CreERT is targeted to the cytokeratin-19 locus, drives TM-dependent recombination in inter- and intralobular ducts . Unlike Muc1IC2, K19 CreERT does not label distal intercalated ducts, and is active in a small fraction of islet cells. Nonetheless, preliminary experiments reported using K19 CreERT provide independent evidence supporting our model: TM treatment at birth results in ≥10% labeling of ducts after one week, but <1% labeling of islets, equivalent to the direct activity of this line in islet cells themselves .
While this manuscript was in preparation, Solar and colleagues  published a study using another exocrine CreERT2 line, driven by the Hnf1β locus. Unlike Muc1IC2, this driver is not active in acini, and labels a higher fraction of duct cells postnatally (approximately 20% at birth and 40% in adults, compared to 10% labeling at either timepoint with Muc1IC2). As with Muc1IC2, lineage-tracing of Hnf1β+ cells revealed duct-to-islet differentiation prior to birth, but none thereafter. Further experiments by these investigators indicate that such differentiation does not occur in the context of injury and regeneration , as previously believed . Our data provide further evidence against postnatal duct-to-islet differentiation in the healthy pancreas, although it remains to be determined if injury can induce neogenesis from Muc1IC2-expressing population.
The Hnf1β-CreERT2 and Muc1IC2 lineage tracing results contradict those obtained with a Cre transgene driven by the Carbonic anhydrase II promoter (CAII-Cre) . Using Rosa26 LacZ reporter mice to detect recombination , these authors report that CAII-Cre drives duct-restricted recombination beginning at E18.5, but labels roughly 15% of β-cells at four weeks of age. We cannot offer an obvious explanation for this discrepancy; given the number of β-cells that we counted, we should have detected such a robust contribution from Muc1IC2-labeled duct cells. One possibility is that CAII-Cre-catalyzed recombination actually begins prior to birth, when Neurog3+ cells are still present [10, 11], but that LacZ expression cannot be detected until one or more days after birth. In fact, half of the newborn pancreata examined in this study already exhibited at least some labeled β-cells (in one pup, as many as 70% of islets contained labeled β-cells), indicating prenatal recombination . These authors have also generated a CAII-CreERT transgene, which could be used to follow postnatal labeling specifically, although their experiments with these mice revealed surprisingly high levels of tamoxifen and Cre-independent LacZ expression in adult islets .
Alternatively, a subpopulation of duct cells with the capacity for islet differentiation might escape labeling by Muc1IC2, K19 CreERT and Hnf1β-CreERT2, but not by CAII-Cre (Figure 11). Indeed, as neither our Cre driver nor those described by others labels a majority of postnatal duct cells [19, 20], the possibility of a substantial unmarked subpopulation is impossible to exclude formally. As three independent and distinct Cre transgenes have yielded identical conclusions, however, the burden of evidence would appear to weigh against postnatal islet neogenesis.
With respect to Muc1IC2 in particular, we note that although its recombination efficiency in utero is even lower than after birth, our experimental approach still identifies islet cells arising from the sparsely labeled embryonic exocrine compartment. Furthermore, we have never observed a Muc1-negative duct cell (Figs. 1, 2, 3, and data not shown), nor is there evidence for anatomical exclusion of Muc1IC2-labeled cells within the ductal network (Figure 6). We also do not observe any obvious change in Muc1 expression or distribution between embryonic stages, when Muc1IC2 does label endocrine cells, and postnatal stages, when it does not. An obvious transition that does occur perinatally is the extinction of Neurog3 expression, which itself weighs against the persistence of duct-to-islet differentiation after birth [10, 11].
From a physiological perspective, it might make sense that expansion of β-cells after birth involves a mechanism independent of neogenesis, as postnatal β-cells must contend with metabolic demands from which embryonic progenitor cells are buffered. In fact, numerous knockout mouse studies indicate the existence of postnatal-specific mechanisms to control β-cell mass [32, 35–39]. Furthermore, recent studies suggest that expansion of β-cell mass in adults, in response to β-cell damage or increased insulin demand, occurs via proliferation rather than neogenesis [13, 37, 40]. Potential exceptions to this rule have been described, including partial pancreatectomy and duct ligation, in which development of new β-cells is accompanied by the re-appearance of Neurog3-expressing cells within the ductal epithelium [16, 17]. Studies using Hnf1β-CreERT2 to mark pre-existing duct cells in such models did not detect contribution to new β-cells, however . The Muc1IC2 line is well suited for similar experiments and, as a tool to mark cells throughout the exocrine pancreas, it should complement and extend results obtained by others.
Our results constitute formal evidence that insulin-producing β-cells, and other endocrine cells of the mature pancreatic islet, derive from ductal cells of the embryonic organ. Furthermore, the ability to trace the lineage of cells expressing Muc1 at different timepoints allows us to compare their differentiation potential before and after birth. We find that Muc1-expressing cells lose the capacity for islet differentiation postnatally, prior to the major increase in β-cell numbers that occurs in juvenile mice. These data add to an emerging model for control of β-cell mass, driven by developmentally-programmed neogenesis in the womb and physiologically-regulated proliferation after birth.
Targeting CreERT2 to the Muc1locus
We followed the procedure of Wu et al.  to generate a Muc1IRES-CreERT2-neo(Muc1IC2neo) targeting vector. In brief, we recombineered a 9.2 kb fragment of the mouse Muc1 gene from a 129Sv BAC library (clone bMQ-356N19, from the Sanger Institute) into a conventional plasmid, introduced an IRES-CreERT2-FRT-neo R -FRT cassette after the endogenous Muc1 stop codon, and flanked the homology arms with thymidine kinase (tk) cassettes (Figure 3D). The targeting vector was then electroporated into R1 ES cells , generously provided by Mario Capecchi, which were selected with G418 and FIAU . 52/96 of the surviving clones exhibited homologous recombination upon Southern blotting with a probe located outside the 5' homology arm. Proper recombination was confirmed for 8/8 of these upon further Southern analyses, and one of these clones, DK5.25 (Figure 3E), was used to generate chimeras (University of Utah, Transgenic Core Facility).
After initial backcrossing to C57BL/6, F1 offspring of a DK5.25 chimera (Figure 3E) were bred to Rosa26 FLPo deletor mice , obtained from the Jackson Laboratory (Bar Harbor, ME), to delete the FRT-flanked neo R cassette. neo R excision yielded the Muc1IRES-CreERT2 (Muc1IC2) allele (data not shown). Multiplex PCR genotyping, producing bands of 357 bp (wildtype) and 464 bp (mutant), was performed using oligos: wt forward: 5'-AATGGCAGTAGCAGTCTCTC-3'; wt reverse: 5'-CACAGCTGGCATAACTAACA-3'; and mutant reverse: 5'-CCACAACTATCCAACTCACA-3'. Muc1IC2 mice were maintained on a CD1 outbred background. Cre reporter mice Rosa26 EYFP  and Rosa26 LacZ  were obtained from the Jackson Laboratory.
Tamoxifen (Sigma T-5648) was dissolved in corn oil, and administered by oral gavage at doses of 5-10 mg to adult mice, or 2 mg by subcutaneous injection of neonates. For timed-pregnancy studies, noon on the day after vaginal plugging was considered embryonic day 0.5 (E0.5). All animal procedures were approved by the Institutional Animal Care and Use Committee.
Immunostaining and analysis
Primary antibodies used in this study
Developmental Studies Hybridoma Bank
Ben Stanger (University of Pennsylvania)
Developmental Studies Hybridoma Bank
Acinar isolation and staining
Acini were isolated by sequential trypsin and collagenase P digestion of minced dorsal pancreas, as described , PFA-fixed for 15 min and adhered to microscope slides by cytospin (Thermo-Fisher). Cytospin slides were stained as per cryosections. For wholemount immunofluorescence of intact tissue, small pieces of the dorsal pancreas were excised and PFA-fixed as above, washed with PBS, permeabilized with 1% Triton X-100 in PBS and stained with primary and secondary antibodies (overnight incubations, followed by extensive PBS/0.1% Tween-20 washes).
List of Abbreviations
Dolichos biflorus agglutinin
enhanced yellow fluorescent protein
green fluorescent protein
internal ribosome entry sequence
We would like to thank Sen Wu, Kirk Thomas and Mario Capecchi for generous gifts of reagents and advice on gene targeting, and Susan Tamowski for blastocyst injections and derivation of chimeric mice. We are grateful to Ben Stanger (University of Pennsylvania) for polyclonal anti-CK19 antiserum. We thank Nadja Makki, Jean-Paul De La O and Kristen Kwan for helpful comments on this manuscript. This work was supported by grants from the Searle Scholars Foundation (06-B-116) and Beta Cell Biology Consortium (U01-DK072473, subaward VUMC35146) to LCM, and a Boehringer Ingelheim Fonds graduate fellowship to DK.
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