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Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors

Department of Molecular and Cellular Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA

View larger version (22K): [in a new window]   Fig. 1. The structure of transgenes (A-C) and the mating scheme (D). The Pdx1Cre transgene has the Cre-coding region directly fused to the SalI-SmaI region of the 5' promoter region of Pdx1. The Pdx1-Cre-ERTM has the Cre-ERTM ATG fused with the Pdx1 ATG (A). The Ngn3-Cre construct directly fuses the Cre-coding region to the 6.5 kb upstream sequence of Ngn3. The Ngn3-Cre-ERTM has the Cre-ERTM ATG fused with the Ngn3 ATG directly (B). Promoters and introns are represented by red lines, untranslated regions by red boxes and coding regions by blue boxes. The transgene polyadenylation signal, which is from the large T antigen polyA region, is noted by black boxes. The yellow and green boxes represent the coding regions of Cre or Cre-ERTM. The reporter used is the Z/AP transgenic line (C) (Lobe et al., 1999). When Pdx1-Cre or Ngn3-Cre strain is crossed with reporters, no further manipulation is needed (D1). When Pdx1-Cre-ERTM or Ngn3-Cre-ERTM is used, tamoxifen (TM) is injected at different embryonic or postnatal days as indicated (D2). HPAP staining was examined 3-8 weeks after birth.

View larger version (115K): [in a new window]   Fig. 2. PDX1+ progenitors give rise to three types of pancreatic tissues in adult mice. Reporter Z/AP mice were crossed with pGK-Cre (B), Pdx1-Cre (C,D) or Pdx1-Cre-ERTM (E-H, tamoxifen was given at E9.5). The pancreata of double transgenic mice were stained for HPAP activity 4 or 8 weeks after birth. (A) HPAP staining is not observed in controls in the absence of recombination. (B) Positive control shows that all pancreatic cells stain for HPAP when a general deletor line, pGK-Cre was mated with the reporter line. (C,D) In pancreata of Pdx1-Cre;Z/AP mice (4 weeks old), all exocrine, endocrine, and duct cells express HPAP, whereas mesenchyme and blood vessels do not. (C) Blood vessels are stained light brown with an anti-mouse PECAM antibody. (D) A confocal image of a Pdx1-Cre;Z/AP pancreas section which is double labeled by ß-galactosidase antibodies (stained red with Cy3) and antibodies against PECAM + vimentin (antigens present in endothelial or mesenchymal cells only, stained green with FITC). Yellow and orange stain (resulting from the combination of red and green) are observed, but not green or red only, demonstrating that only the blood vessel or mesenchymal cells maintain lacZ expression. Note that this section was not stained for HPAP, i.e. the dark areas are acini, islets (i) and ducts. (E-H) The HPAP staining pattern (8 weeks old) when Pdx1-Cre-ERTM animals are crossed with Z/AP and TM is administered at E9.5. Islet (E), acini (F) and ducts (G-H) are labeled in a mosaic fashion. In H, the small ducts are stained with Wisteria floribunda agglutinin as brown and HPAP as blue. Note the blue staining in the small duct but not the adjacent acinar cells. (a, acini, broken white lines; d, duct, broken green lines; i, islets, broken red lines; v, blood vessel). Scale bars: in D, 20 µm; in G, 20 µm; in H, 20 µm for A, B, C, E, F and H.

View larger version (161K): [in a new window]   Fig. 3. Duct progenitors express Pdx1 in the period between E9.5-E12.5. Tamoxifen is administered at different stages of development and the HPAP expression in 8-week-old double transgenic animals is determined (when TM is administered to 8-week-old animals, the HPAP staining is performed 3 days later). (A,B) Acini and islet staining when TM was injected at E8.5. In B, the duct is stained brown with HRP-conjugated Wisteria floribunda agglutinin. Note that the brown duct is not blue, whereas acinar cells surrounding the duct are HPAP+ and stain blue. (C) HPAP+ cells within ducts (d), islets (i) and acinar structures when TM was injected at E10.5. (D-F) Acini and islet staining when TM was administered at E12.5 (D), 3 weeks (E) or 8 weeks (F) after birth. (a, acini; d, duct, broken green lines; i, islets, broken red lines; blood vessel, broken black lines.) Scale bars: in B, 20 µm; in F, 20 µm for A, C, D, E and F.

View larger version (23K): [in a new window]   Fig. 4. Relative frequency of duct cell progenitors. Eight-week-old pancreata that received tamoxifen at different stages of development were stained for HPAP and counted (see Materials and Methods). Raw numbers are shown in A. The histogram (B) shows the relative frequency of HPAP+ cells in exocrine (green), islet (blue) or duct (red) tissue. Variations in HPAP labeling efficiency induced by TM at different stages were normalized to the exocrine tissue.

View larger version (142K): [in a new window]   Fig. 5. NGN3+ cells form islets. Correct expression of the Ngn3 promoter-driven transgene is assessed by in situ hybridization on adjacent sections. (A,B) Adjacent sections of an E13.5 transgenic pancreas probed with Ngn3 (A) or Cre-ERTM (B) cRNA. To study the lineage of the Ngn3-expressing cells, TM is given to embryos at different stages and the pancreata of double transgenic animals are examined for HPAP expression or co-expression of HPAP with various hormones at 3-8 weeks of age. (C,D) HPAP staining in islet cells when TM was injected at E8.5 or E12.5, respectively (8-week-old pancreata are shown). (E) A confocal image that shows hormone (Ins, Glu, Som, PP) and HPAP double-positive cells (arrows) when TM was injected at E8.5. Each picture is a single scan. Because HPAP is a membrane-bound protein while the hormones are cytoplasmic proteins, we do not always see colocalization of HPAP and hormone, as in HPAP/PP staining (i, islets, broken red lines). Scale bars: in B, 25 µm for A,B; in D, 25 µm for C,D; in E, 5 µm.

View larger version (169K): [in a new window]   Fig. 6. NGN3+ cells contribute to the renewal or maintenance of islets in adult mice. Tamoxifen was injected into 3- or 8-week-old Z/AP;Ngn3-Cre-ERTM animals and HPAP expression was examined 1, 3, or 7 days later. (A) A control showing that no HPAP+ cells are detected in an 8-week-old Ngn3-Cre-ERTM; Z/AP pancreas without TM injection. (B) HPAP+ cells detected in islets 1 day after TM was injected into an 8-week-old mouse. (C) An enlarged area of B. (D) HPAP+ cells in islets 7 days after TM injection. In no case were HPAP+ cells observed in non-islet structures (d, duct, broken green lines; i, islets, broken red lines). Scale bars: in B, 25 µm for A,B; in D, 25 µm for C,D.

View larger version (183K): [in a new window]   Fig. 7. Duct cell progenitors do not express Ngn3. Ngn3-Cre transgenic animals are crossed with the Z/AP reporter line and HPAP expression in pancreas is characterized in postnatal mice (A,B) or embryos (C,D). (A,B) In 1- or 8-week-old animals, only islet cells express HPAP (i, islet, broken red line), and no duct cells express HPAP (d, duct, broken green line). (C) In E14.5 pancreas, HPAP+ cells reside in duct-like structures (dl, duct-like structures, broken green line), as identified by the presence of a hole. Yet, these HPAP+ cells are endocrine progenitors (ep, red arrow). (D) At E16.5, the HPAP+ cells or endocrine progenitors (ep, broken red lines) form cord-like or islet-like structure and some duct-like structures that do not express HPAP (dl, broken green lines) are also observed. Scale bar: 25 µm.

View larger version (163K): [in a new window]   Fig. 8. The progeny of NGN3+ cells are mitotically active at E14, but they suppress Ngn3 expression. Ngn3-Cre-ERTM; Z/AP embryos were given TM at E8.5-E10.5 and the mitotic status of HPAP+ cells was assayed by BrdU incorporation. Ngn3 expression and the differentiation of these HPAP+ cells was examined between E13.75 and E14 using NGN3 or NEUROD antibodies. (A) A clone composed of three HPAP+ cells (arrow), indicating that they are derived from a cell that expressed Ngn3. (B) An adjacent section to that in A shows these HPAP+ cells have incorporated BrdU. (C) HPAP staining (blue) and NGN3 protein staining (brown; with a polyclonal Ab). The cell indicated by the red arrowhead does not stain for NGN3 protein, whereas two other cells (black arrowheads) in this section are NGN3+. (D) An HPAP+ cell (blue stain, red arrowhead) that has differentiated and is expressing NEUROD protein (brown antibody stain). Other cells in this field are not HPAP+ but do stain for NEUROD. Scale bar: 8 µm.

View larger version (21K): [in a new window]   Fig. 9. Two models for the lineage of exocrine, endocrine and duct cells. In both models, the pancreatic duct progenitors only express Pdx1 from E9.5 to E12.5. In Model 1, the duct lineage diverged from that of the endocrine/exocrine cells before E8.5. The endocrine and exocrine cells are distinguished by the presence of Ngn3 expression. In Model 2, the duct and endocrine/exocrine lineage diverged between E9.5 and E12.5. In the later model, suppressive or inductive signals are needed to allow for the development of duct fate.