Neurogenin 3+ cells contribute to β-cell neogenesis and proliferation in injured adult mouse pancreas

Abstract

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to β cells ex vivo. Here we evaluate the role of Ngn3(+) cells in β cell expansion in situ. PDL not only induced doubling of the β cell volume but also increased the total number of islets. β cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the β cell expansion was attributable to proliferation of pre-existing β cells. At sufficiently high Ngn3 expression level, upto 14% of all β cells and 40% of small islet β cells derived from non-β cells. Moreover, β cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing β cells supporting a key role for Ngn3(+) insulin(-) cells in β cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed β cells as well as reprogramming of non-β cells contribute to in vivo β cell expansion in the injured pancreas of adult mice.

Figure 1

PDL induces islet formation and β cell proliferation in adult mouse pancreas. Eight-week-old male Balb-c mice, underwent PDL or Sham surgery. (a) PDL and Sham tail total β cell volume (mm³) and (b) number of β cell clusters in the indicated diameter (μm) categories (n=3; *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, PDL versus Sham tail). (c) Panels show the total tissue volume (gray) and insulin⁺ volumes (red) detected by OPT microscopy in tail regions of Sham or PDL operated pancreases. The total islet volume increased after PDL to 2.0 × 10⁹±3.5 × 10⁸ μm³ (compared with Sham tail 1.1 × 10⁹±2.8 × 10⁸ μm³; n=3; P=0.056). The total number of insulin⁺ islets significantly increased from 1000±184 in Sham to 1539±77 in PDL tails (n=3; P<0.05). This increase could be attributed to a significant increase in small islets (<10⁶ μm³) (711±107 islets in Sham versus 1082±25 islets in PDL; n=3; P<0.05). (d,e) β cell proliferation was determined in ligated PDL tail, unligated PDL head or Sham tail pancreas, at 7 and 14 days post-PDL by immunofluorescence staining (d) for insulin and Ki67 (n=5–14) or (e) for insulin, Nkx6.1 and Ki67 (n=3) (***P<0.001, ns: P>0.05, versus Sham tail or PDL head by two-way ANOVA). (f) 5-iodo-2′-deoxyuridine (IdU, 1 mg/ml) was i.p. injected precisely at 8 and 4 h before sacrifice at the indicated time points following PDL. The percentage of IdU-labeled β cells was determined by immunostaining for insulin and IdU (n=3, *P<0.05, **P<0.01 versus Sham tail by two-way ANOVA). 1800±200 β cells were counted per tissue sample. See also Supplementary Figure S4A

Figure 2

β cells of PDL pancreas redivide without extended delay (‘refractory period‘). Insulin⁺ β cells were scored for CldU and/or IdU labeling using analog-specific antibodies. By labeling a first cell division with CldU (blue) and a second one with IdU (green), sequential divisions result in colabeled β cells (blue and green). (a) Neonatal Balb-c mice received i.p. CldU on day 5 (P5), and i.p. IdU on postnatal day 6 (P6), and were sacrificed 24 h later. P6 mice pancreas displayed high percentages of CldU-single positive (blue nuclei) and IdU-single positive (green nuclei) β cells (red) but only very few double-labeled CldU⁺IdU⁺ β cells (blue and green nuclei). (n=6, 7316 insulin⁺ cells evaluated). Eight-week-old male Balb-c mice underwent PDL surgery and CldU and IdU were sequentially administered via the drinking water: (b) 3 days CldU+3 days IdU (n=6, 8172 β cells counted), (c) 7 days CldU+7 days IdU (n=5, 5505 β cells counted), (d) 7 days CldU+28 days IdU (n=3, 5816 β cells counted). (e) On the basis of the data obtained in (a–d), the likelihood of redivision of β cells was calculated (see formula) and plotted versus duration of the cognate IdU labeling period, fitting the modeled exponential function f(x)=(1-a^x-r)/(1-a^x), where x=days of IdU labeling, a=probability of non-labeling per day and r=refractory period in days. The curve fitted the experimental PDL data (b–d), as well as data obtained by for partial pancreatectomy (PPx), pregnant mice (Preg), neonatal mice (Neo), or non-treated mice (non-treated). For comparison, our data on neonatal mice (a) are also represented by a datapoint (*) in (e). See also Supplementary Figures S2 and S3. Scale bars represent 50 μm

Figure 3

Small β cell clusters are the preferred site of β cell (re)division in PDL pancreas. Eight-week-old male Balb-c mice underwent PDL surgery and were continuously labeled with thymidine analogs via the drinking water. Proliferation was analyzed by immunofluorescence staining for either IdU or IdU and CldU in insulin⁺ cells. Small clusters were defined as clusters of 1–20 β cells on cross-section while large clusters contained 21–200 β cells. Data are expressed as mean±S.E.M. of the percentage of β cells that were positive for thymidine analog in each size category. (a) Seven days IdU-labeling of β cells in PDL tail (n=5, **P<0.005), (b) 14 days IdU-labeling of β cells in PDL head and tail (n=4–7, *P<0.02, ns: P>0.05). (c) Thirty-five days of IdU-labeling of β cells in the PDL tail (n=3, **P<0.01). (d) Data from (c) analyzed for subcategories of clusters, and expressed as the percentage of clusters consisting of only analog⁺ β cells. Clusters comprising >60 insulin⁺ cells never consisted entirely of analog⁺ β cells. (n=3, **P<0.01 by one-way Anova) (e) 7 days CldU labeling followed by 28 days IdU labeling and evaluation of the percentage of CldU⁺IdU⁺ labeled (redivided) β cells according to cluster size (n=3, **P<0.01 by one-way Anova). (f) 8 h IdU pulse labeling of β cells in small and large clusters of PDL and Sham tail pancreas (n=3; *P<0.05, **P<0.01 versus same time point for Sham tail, by 2-way Anova). See also Supplementary Figure S4B

Figure 4

β cells derived from Ngn3⁺ cells preferentially locate and proliferate in small β cell clusters of PDL pancreas. (a) Description of the transgenic mouse model and the labeling strategy for heritable tracing of Ngn3 expressing cells. Ngn3=Ngn3 gene promoter, R26=Rosa26 locus, loxP=loxP sequence, STOP=transcriptional STOP sequence, YFP=yellow fluorescent protein. Tamoxifen injection (TAM, *4 mg/injection) of bigenic Ngn3^CreERT;R26^YFP mice results in Cre-mediated removal of the lox-P flanked STOP sequence, and permanent expression of the YFP reporter gene in cells with an active Ngn3 promoter and their descendants. Eight-week-old male bigenic mice underwent PDL surgery and were TAM-injected (s.c., starting on day of surgery: 4 mg TAM, repeated once every-other-day, five injections in total). (b) YFP-staining in small clusters of Insulin⁺CK19⁺ cells associated with CK19⁺ duct cells of PDL tail. (c) YFP⁺INS⁺ β cells in islets of PDL tail. (d) Quantification of the percentage YFP⁺ β cells in PDL head and tail with the indicated range of Ngn3 mRNA expression level in PDL tail 14 days after ligation (n=3, *P<0.05, **P<0.01 by two-way ANOVA; a total of 21295 β cells evaluated). Ngn3 mRNA was expressed relative to cyclophilin A mRNA and normalized to Ngn3 expression in duodenum. (e) Quantification of the percentage of YFP⁺ β cells in small (1–20 insulin⁺ cells) and large (21–200 insulin⁺ cells) clusters in PDL tail day 14 and 35 (three mice per experimental condition, ***P<0.001 versus large clusters, by two-way ANOVA). (f) Quantification of percentage IdU-labeling (proliferation) in YFP⁺ β cells in small and large clusters (n=3, *P<0.05, versus large clusters). (See also Supplementary Figure S5). Scale bars represent 50 μm

Figure 5

New β cells can derive from non-β cells in PDL pancreas. (a) Description of the transgenic mouse and the strategy for labeling and tracing pre-existing β cells (legend as in Figure 4). Tamoxifen s.c. injection (*TAM, 4 mg/injection) of bigenic RIPCre^ERT;R26^lacZ mice of 8 weeks results in transient nuclear translocation of CreERT, leading to removal of the loxP-flanked STOP sequence and permanent heritable expression of the lacZ reporter gene (encoding β galactosidase, bGal) in β cells. A 14 days TAM-washout (WO) period assured the absence of residual TAM at the time of surgery. PDL-surgery was followed by a label-chase period of 14 or 35 days after which islets in PDL head or PDL tail were examined by immunostaining for bGal expression in insulin⁺ cells. Non β cell-derived β cells would dilute the label and decrease the preponderance of bGal⁺ insulin⁺ cells. (b) bGal expression in insulin⁺ cells following 14 or (c) 35-days chase period. Left panels, preponderance of bGal⁺ insulin⁺ cells; right panels, preponderance of bGal⁺ insulin⁺ cells in clusters of different sizes. Data are expressed as the mean±S.E.M. obtained with three mice (*P<0.05, **P<0.01). (d) Immunofluorescent staining for detection of pre-existing (bGal⁺ insulin⁺) β cells in PDL tail and (e) in PDL head, after 35 days chase period. (See also Supplementary Figures S4C,S6 and S7). Scale bars represent 50 μm

Figure 6

Ngn3⁺ cells are required while Ngn3 in pre-existing β cells is dispensable for the β cell proliferation in PDL pancreas. (a) Description of the transgenic mouse model and strategy used for selective ablation of Ngn3⁺ cells by DT. Tamoxifen injection in bigenic Ngn3Cre^ERT−/+;R26^iDTR+/+ mice results in permanent heritable expression of the DTR gene in Ngn3⁺ cells and cells derived thereof. Eight-week-old bigenic mice and monogenic Ngn3Cre^ERT−/−;R26^iDTR+/+ littermates underwent PDL surgery, s.c. TAM administration (*TAM, 4 mg/injection), and i.v. DT (# daily injections, 125 ng/injection). (b) Ngn3 mRNA expression measured by qRT-PCR in duodenum, and PDL head and tail pancreas of mono- (n=3) or bigenic mice (n=4; *P<0.05 versus the monogenic tissue). Data were normalized to cyclophilin A mRNA and expressed relative to the Ngn3 level in monogenic duodenum. (c) Total insulin content (μg) of PDL head and tail pancreas of mono- or bigenic mice (n=3, **P<0.01). (d) Percentage of Ki67⁺ β cells in PDL head and tail pancreas of mono- (n=3) or bigenic (n=4) mice (**P<0.02). (e) Percentage of Ki67⁺ β cells in small (1–20) or large (21–200) cell clusters of PDL tail (n=3–4, *P<0.05, ns: P>0.05 by two-way ANOVA). Data are expressed as the mean±S.E.M. See also Supplementary Figure S8. (f) Following TAM injection and wash out, RIP^CreERT;Ngn3^+/+ and RIP^CreERT;Ngn3^lox/lox mice underwent PDL surgery. Immunofluorescence staining for Ki67 and insulin in PDL tail tissues is shown. β cell proliferation was quantified and expressed as the mean±S.E.M. (n=6, ns: P>0.05; a total of 30250 β cells were evaluated). Scale bar represents 50 μm. (g) RIP^CreERT;R26^YFP;Ngn3^+/lox mice were treated with TAM as in (f). Islets were isolated and dissociated to sort the islet cells on the basis of TSQ (Dapi, not shown) and YFP fluorescence (FITC). The window (left panel) comprises all the TSQ^high (endocrine) cells. The gating indicates YFP⁺ β cells (green dots). YFP⁻ islet cells (red dots) were also collected. YFP⁻ islet cells had a wild-type (Ngn3⁺) and a floxed (Ngn3^lox) allele, whereas YFP⁺ β cells had lost the Ngn3^lox allele. PCR results for two separate isolations are shown (right panel)