Preexisting pancreatic acinar cells contribute to acinar cell, but not islet β cell, regeneration (original) (raw)
We generated a transgenic mouse in which a TAM-inducible Cre recombinase (CreERT2, which encodes a Cre recombinase [Cre] fused to a mutant estrogen receptor ligand-binding domain [ERT2] that is selectively responsive to TAM) is regulated by the strong and highly acinar cell–specific elastase I promoter (ElastaseCreERT2 mouse; Figure 1A) (16) in order to facilitate lineage tracing studies and experiments involving inducible and selective gene inactivation in pancreatic acinar cells. Two founders were identified, one of which expressed Cre recombinase, as detected by RT-PCR of pancreatic RNA (Figure 1C). A tissue distribution experiment revealed appropriate restriction of transgene expression to the pancreas (Figure 1C). Notably, no substantial Cre recombinase mRNA expression was detected in the stomach, small or large intestine, heart, lungs, liver, kidney, or spleen. This is consistent with the faithful pancreas-specific expression directed by this promoter fragment previously reported in other transgenic models (17, 18).
CreERT2 expression is restricted to the pancreas of ElastaseCreERT2 transgenic mice. (A) Schematic depiction of the elastase CreERT2 transgene. The transgene contains the 0.5-kb proximal rat elastase I promoter (Elas; gray), the rabbit β-globin intron (RBG intron; black), the coding region for CreERT2 (white), and the rabbit β-globin polyadenylation cassette (RBG PolyA; black). (B) Schematic depiction of the Rosa26r reporter locus and the effect of TAM-induced Cre recombinase activity to excise a loxP-flanked stop sequence (dashed lines), thereby allowing transcription of LacZ (i.e., β-galactosidase; arrow). (C) Tissue distribution of Cre recombinase expression, determined by RT-PCR for Cre recombinase mRNA using total RNA isolated from the designated tissues of 16-week-old transgenic mice. Panc, pancreas; Stom, stomach; SI, small intestine; LI, large intestine; Ht, heart; Lu, lung; Li, liver; Kid, kidney; Spl, spleen. (D) Time course indicating low basal Cre-mediated recombination and high inducibility by TAM administration. RT-PCR for β-galactosidase mRNA carried out using RNA isolated from pancreata of 16-week-old transgenic mice left untreatd or after 3, 7, or 10 days of TAM treatment.
To determine the efficiency and specificity of Cre-mediated recombination in ElastaseCreERT2 mice, we crossed ElastaseCreERT2Tg/+ mice with Rosa26r reporter mice (19) to generate ElastaseCreERT2Tg/+Rosa26r+/– mice. Expression of β-galactosidase was restricted to cells that underwent Cre-mediated excision of an upstream stop cassette in the LacZ-engineered endogenous Rosa locus, as well as the progeny of these cells (Figure 1B). To determine the levels of basal and TAM-induced Cre-mediated recombination, RT-PCR for β-galactosidase mRNA was carried out using RNA isolated from the pancreata of ElastaseCreERT2Tg/+Rosa26r+/– mice left untreated or treated for 3, 7, or 10 days with TAM. As shown in Figure 1D, no β-galactosidase mRNA was observed in untreated mice, indicating no detectable basal Cre-mediated recombination in the absence of TAM treatment. Recombination was detected by 7 days of treatment, and higher levels of recombination were observed after 10 days of treatment.
To determine whether transgene expression is restricted to the acinar lineage of the pancreas, ElastaseCreERT2Tg/+ mice were initially treated with TAM for 3 days or left untreated. Pancreatic sections were visualized for immunoreactivity associated with Cre recombinase as well as insulin, a key marker of the endocrine pancreatic β cell that constitutes the majority of cells in the islets of Langerhans. In the control pancreas, Cre immunoreactivity was localized to the cytoplasm in a subset of pancreatic cells. After 3 days of TAM treatment, nuclear Cre was observed in a mosaic pattern that was restricted to the exocrine portion of the pancreas (data not shown). Cre recombinase expression was not observed in islets of the endocrine pancreas. After 21 days of TAM treatment delivered by subcutaneous continuous delivery pellet, β-galactosidase was visualized in approximately 30% of acinar cells in a patchy but somewhat lobular pattern, reflecting the mosaic expression pattern of Cre recombinase (Figure 2, B–D), as has been frequently observed with other Cre recombinase driver lines (20). In the absence of TAM administration, very few acinar cells expressed β-galactosidase, indicating tight regulation of Cre activity by TAM (Figure 2A). In TAM-treated mice, no β-galactosidase–positive cells were detected within islets at any time point. Furthermore, double staining with biotinylated dolichos biflorus agglutinin (DBA) lectin (which selectively recognizes ductal epithelial cells; refs. 21, 22, and data not shown) or with ductal-specific cytokeratin 19 showed complete absence of labeling in all classes of ductal epithelial cells, including the specialized centroacinar cell (Figure 2, E–N). Thus, the elastase CreERT2 transgene is expressed only in the exocrine pancreas, and induction of recombinase activity in adult mice appears to induce loxP rearrangement selectively in acinar cells.
Cre-mediated recombination is restricted to the acinar cell lineage in TAM-treated ElastaseCreERT2Tg/+Rosa26r+/– mice. ElastaseCreERT2 mouse pancreata from 16-week-old animals stained with X-gal (A, B, F–H, J, and K), anti–Cre recombinase antiserum (red stain; C, L, and N), anti-amylase (green stain; D), or anti–cytokeratin 19 (red stain in E, G–I, and K; green stain in M and N) without TAM treatment (A) and after 21 days of TAM treatment (B–N). Arrowheads indicate centroacinar cells identified by cytokeratin-19 immunoreactivity (I and M) but no β-galactosidase activity (J) or Cre recombinase immunoreactivity (L). Original magnification, ×200 (A–H); ×400 (I–K); ×1,000 (L–N).
Once characterized, ElastaseCreERT2Tg/+Rosa26r+/– mice were used in lineage tracing studies to determine the transdifferentiation potential of acinar cells in vivo. In control, sham-operated mice, the islet lineage was not labeled during observation periods of up to 6 weeks after the completion of TAM administration (Figure 3), indicating that adult acinar cells do not serve as islet progenitor cells during normal pancreas function in mice. To determine whether acinar-to-islet transdifferentiation occurs under conditions in which islet regeneration is provoked, we performed 70%–80% partial pancreatectomy (Ppx) or sham operation on ElastaseCreERT2Tg/+Rosa26r+/– mice and then treated them with either vehicle or exendin-4 (Ex-4) to further promote β cell regeneration (23). We confirmed β cell mass regeneration by quantitation at 1 month after surgery, when β cell mass in the Ppx remnant had recovered to 73% that of the sham-operated group (P = NS; Figure 4B), indicating a robust regeneration of β cell mass similar to our previous observations in CD1 mice (24). The exocrine portion of the pancreatic remnant only partially recovered, as indicated by reduced wet weight compared with the pancreata of sham-operated controls (Figure 4C). The biologic efficacy of Ex-4 was demonstrated by improved glucose tolerance at 2 weeks in Ex-4– versus vehicle-treated Ppx mice (Figure 4A).
No evidence of acinar-to-islet transdifferentiation after Ppx. ElastaseCreERT2Tg/+Rosa26r+/– mice were treated with TAM for 3 weeks by subcutaneous continuous delivery pellet and subjected to 80% Ppx or sham surgery after a 1-week washout period. After surgery, mice were randomized to receive daily injections of vehicle (Veh) or Ex-4 (1 nmol/kg body wt i.p.; n = 4–5 per group). Tissues were harvested 2 weeks later and stained for β-galactosidase activity (blue stain, left panels) and insulin (red stain, middle panels). Merged images are shown in the right panels. Sections depicted are from 16-week-old mice. Additional experiments carried out in mice ranging in age from 12 weeks to 6 months failed to reveal evidence of acinar-to-islet transdifferentiation. Original magnification, ×200.
Recovery of β cell mass after Ppx and biological efficacy of Ex-4 treatment. (A) Glucose tolerance tests were performed i.p. 2 weeks after Ppx or sham surgery in mice treated with vehicle or Ex-4 (n = 4–5 per group). Black line denotes sham surgery plus treatment with vehicle, blue dashed line denotes Ppx plus treatment with vehicle, gray line denotes sham surgery plus treatment with Ex-4, and dashed black line denotes Ppx plus treatment with Ex-4. P < 0.05, Ppx plus vehicle versus all other groups; ANOVA. (B) Mass of β cells (in mg) was determined by point counting morphometry 4–5 weeks after surgery. (C) Weight (in g) of the pancreatic remnant at 4–5 weeks after surgery.
At no time did we observe β-galactosidase labeling within pancreatic islets, indicating that acinar cells do not serve as islet progenitors during regeneration after Ppx. Furthermore, Ex-4 does not promote acinar-to-islet transdifferentiation in vivo, in contrast to its ability to promote transdifferentiation of acinar tumor AR42J cells into insulin-expressing β-like cells in culture (14). Similarly, no β-galactosidase–labeled ductal epithelial cells were observed in vivo, in contrast to our previous observations of acinar-to-ductal transdifferentiation when TAM-treated ElastaseCreERT2Tg/+Rosa26r+/– acini are maintained in culture in the presence of TGF-α (8). Taken together, these data indicate that acinar cells may have limited transdifferentiation potential in vivo.
In contrast, our present studies clearly indicate that preexisting acinar cells serve as progenitors for newly formed acinar cells during regeneration, similar to recent reports of β cell replication as the primary mechanism for postnatal β cell replacement and during adult islet regeneration (25, 26). In order to distinguish between preexisting and newly generated cells, BrdU was administered for 1 week beginning immediately after surgery and continuing until tissue harvest. BrdU was found to be incorporated into patches of acinar cells distributed throughout the acinar parenchyma, but not restricted to individual lobes (Figure 5A). We observed BrdU incorporation into β-galactosidase–positive and –negative cells, indicating that β-galactosidase–expressing cells contribute to acinar cell replacement (Figure 5B). To determine whether an unlabeled progenitor cell type, as opposed to differentiated acinar cells, markedly contributes to the formation of new acinar cells, we assessed the extent of β-galactosidase activity in these areas of high proliferation. If an unlabeled progenitor cell type contributes to acinar cell regeneration, then a decrease in the percentage of cells expressing the β-galactosidase label should be observed, such that proliferative areas are those with low proportions of cells expressing β-galactosidase. On the other hand, if acinar cell regeneration occurs primarily through replication of preexisting acinar cells, no decrease in labeling would be expected. In fact, we observed that high levels of BrdU incorporation occurred with equal frequency in areas of high, medium, and low β-galactosidase expression (Figure 5C), arguing against a contribution from a β-galactosidase–negative progenitor cell type. Because the specialized centroacinar cell is not labeled in this model, the results also argue against a role for centroacinar cells as progenitors for acinar cell regeneration after Ppx. In agreement with previous studies demonstrating the importance of postnatal β cell replication (25, 26), we observed robust incorporation of BrdU within regenerating islets after Ppx (Figure 5D).
Regeneration of acinar cells from preexisting acinar cells after Ppx. (A) BrdU incorporation (brown stain) and β-galactosidase activity (blue stain) were assessed in ElastaseCreERT2Tg/+Rosa26r+/– mice that were continuously exposed to BrdU in their drinking water beginning immediately after surgery until tissue harvest 1 week later. (B) Magnification of boxed area in A. The white arrow denotes a BrdU-positive and β-galactosidase–positive acinar cell; the black arrow denotes a BrdU-positive and β-galactosidase–negative acinar cell. (C) Percent of fields with high levels of BrdU incorporation segregated according to the proportion of acinar cells expressing β-galactosidase (low, 0%–30%; medium, 30%–50%; high, 50%–100%). Quantitation of BrdU incorporation as a function of β-galactosidase expression was performed as described in Methods. (D) BrdU incorporation within a representative islet after Ppx. Original magnification, ×200 (A and D); ×400 (B).
Finally, we examined acinar cell transdifferentiation potential in 2 other models of pancreatic injury in order to determine whether the ability of acinar cells to serve as islet progenitors is model specific. Pancreatitis was induced by a 2-day course of cerulein injections that resulted in marked exocrine atrophy and inflammatory infiltration (Figure 6, A and B) associated with apoptosis and dedifferentiation (27). The impressive degree of injury was nearly completely reversed over a 7-day period (Figure 6C). Jensen et al. reported peak activation of Notch1/2 after 3 days and a reexpression of high levels of PDX-1, suggesting reversion of injured cells to a progenitor-like state (27). However, we again found no β-galactosidase labeling in the endocrine lineage, indicating that these cells do not serve as islet progenitors even after Ex-4 administration during the peak period of Notch expression (Figure 6, D–F). In agreement with Jensen et al. (27), we did not observe labeled cells in the ductal epithelium, suggesting that acinar-to-ductal transdifferentiation did not occur in this model. Furthermore, using a lineage tracing approach similar to ours, minimal decrease of labeled acinar cells was found after recovery from cerulein-induced pancreatitis, again indicating regeneration primarily from preexisting acinar cells (J. Jensen, unpublished observations). Finally, we did not observe acinar-to-islet transdifferentiation in the pancreatic duct ligation model, although the characteristic metaplastic epithelium distal to the ligation was marked by β-galactosidase activity, indicating an acinar cell of origin (data not shown).
No evidence of acinar-to-islet transdifferentiation after cerulein-induced pancreatitis. ElastaseCreERT2Tg/+Rosa26r+/– mice were treated with TAM for 3 weeks by subcutaneous continuous delivery pellet and subjected to a 2-day course of serial cerulein injections as described in Methods. (A–C) Severe exocrine inflammation but intact islets were evident 1 day after the initiation of treatment (A and B), with histologic recovery by 7 days (C). Cerulein-treated mice were randomized to receive daily injections of vehicle or Ex-4 (1 nmol/kg body wt i.p.) on day 3, the time of maximal Notch activation. (D–F) Tissues were harvested on day 7, and β-galactosidase activity (blue stain) and insulin immunoreactivity (red stain) were visualized. (D) Pancreas from a control mouse that received only PBS vehicle. (E and F) Pancreata from cerulein-treated mice that received vehicle (E) or Ex-4 (F). Original magnification, ×200.