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First published online 28 September 2005
doi: 10.1242/dev.02063

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ß-Catenin is essential for pancreatic acinar but not islet development

L. Charles Murtaugh^*, Anica C. Law, Yuval Dor and Douglas A. Melton

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

View larger version (85K): [in a new window]   Fig. 1. Potential mediators of Wnt signaling in the developing pancreas. (A,B) In situ hybridization (purple) to E13.5 pancreatic primordia detects co-expression of Pdx1 (A) and the ß-catenin-binding transcription factor Tcf4 (B), while the related factor Lef1 is not expressed (data not shown). (C-D') To confirm the specificity of pan-specific (C,D) and dephospho-specific (C',D') anti-ß-catenin monoclonal antibodies, we immunostained (brown) near-adjacent sections of embryonic spinal cord and neonatal (P0) intestine. As expected, anti-dephospho-ß-catenin recognizes regions where Wnt signaling is known to be active, such as the ventricular zone of the spinal cord (arrow) and the intestinal crypts (arrowhead). (E-I') Pan-ß-catenin (E-I) and dephospho-ß-catenin (E'-I') staining was similarly performed on various stages of developing pancreas. Arrowheads and arrows indicate morphologically recognizable islet and acinar tissue, respectively. Dephospho-ß-catenin is specifically detected in the early pancreatic epithelium, and declines as the organ matures. me, pancreatic mesenchyme; ep, pancreatic epithelium.

View larger version (65K): [in a new window]   Fig. 2. Pancreas-specific ß-catenin knockout (PBKO) mice. (A) Breeding scheme to generate wild-type (green) and PBKO (red) genotypes. (B,C) Gross appearance of neonatal wild-type and PBKO guts. (D,E) Dorsal (red) and ventral (blue) pancreata highlighted in above genotypes. (F,G) Hematoxylin and Eosin stained sections of wild-type and PBKO pancreata reveals the abnormal morphology of latter genotype, and the scarcity of acini (arrowheads). (H-K) Immunostaining (brown) for insulin (H,I) and glucagon (J,K) in wild-type and PBKO pancreata reveals large clumps of islet tissue in the latter genotype; Hematoxylin counterstain is blue.

View larger version (44K): [in a new window]   Fig. 3. Maintenance of early pancreatic progenitors in the absence of ß-catenin. Three-color confocal immunofluorescence for ß-catenin (A,A'), Pdx1 (B,B') and glucagon (C,C') in E11.5 dorsal pancreatic buds. The relative distribution of Pdx1+ and glucagon+ cells is normal in regions of the PBKO pancreas lacking ß-catenin (A'-D'). Immunohistochemical staining for pan-ß-catenin or Pdx1 in near-adjacent sections of E13.5 wild-type (E,F) or PBKO (E',F') pancreata reveals normal expression of Pdx1 in ß-catenin-deficient epithelia. Pancreata are counterstained with Hematoxylin (blue) in F,F'. Arrowhead in E' indicates background staining that outlines ß-catenin-deficient epithelia.

View larger version (93K): [in a new window]   Fig. 4. Impaired proliferation in ß-catenin knockout pancreata. (A-E') E11.5 wild-type (A-E) or PBKO (A'-E') dorsal pancreata were analyzed by immunohistochemistry or in situ hybridization. Anti-BrdU staining (brown, A,A') labels proliferating cells; pregnant females were injected 1 hour prior to harvest (50 µg/kg). PBKO pancreata show a moderate but significant decrease in proliferation (35.4% BrdU+) compared with wild type (44.5% BrdU+). In situ hybridization (purple) reveals little or no change in expression of the potential Wnt/ß-catenin target genes cyclin D1 (B,B') and Id2 (C,C') between wild type and PBKO, whereas that of Myc (D,D') is strongly downregulated in PBKO. Expression of the Notch target gene Hes1 (E,E') is similar in the two genotypes. Red dashed lines outline pancreatic epithelia.

View larger version (37K): [in a new window]   Fig. 5. Relative impact of ß-catenin deletion on the endocrine and exocrine compartments. In situ hybridization (purple) to detect insulin1 (A,A'), proglucagon (B,B') and carboxypeptidaseA1 (Cpa1) (C,C') on near-adjacent sections of E15.5 wild-type (A-C) or PBKO (A'-C') pancreata. (D) Morphometric quantitation of in situ hybridization to calculate endocrine and exocrine volumes in wild-type (n=3) and PBKO (Catnb/lox;Pdx1-Cre; n=4) pancreata. Shown are individual measurements (points) and means (bars); the difference between wild type (green) and PBKO (red) is statistically significant only for Cpa1 (P<0.05 by two-tailed t-test).

View larger version (100K): [in a new window]   Fig. 6. Relative requirement for ß-catenin in individual endocrine and exocrine cells. Confocal immunofluorescence on E15.5 (A-I') or neonatal (J-L') wild-type versus PBKO pancreata, to detect co-expression of ß-catenin (A,A',D,D',G,G',J,J') with insulin (B,B'), glucagon (E,E'), amylase (H,H') or the duct marker DBA lectin (K,K'). Filled arrowheads indicate examples of cells co-expressing the markers; open arrowheads indicate a lack of co-expression. Note that all insulin+ and glucagon+ cells are ß-catenin– in PBKO sections (C',F'), as are the majority of DBA+ cells (L'). By contrast, all residual amylase+ cells in these pancreata are ß-catenin+ (I'), indicating that they derive from cells in which the Catnb gene did not undergo Cre-mediated excision.

View larger version (119K): [in a new window]   Fig. 7. Loss of Ptf1a/p48+ acinar precursors in the absence of ß-catenin. Confocal immunofluorescence at E13.5 detects peripheral clusters of Ptf1a+ cells in wild-type pancreata (A-C), presumably representing acinar precursor cells. Ptf1a+ cells are much more infrequent in PBKO pancreata (A'-C'), and are restricted to residual ß-catenin+ regions, indicating that they derive from cells in which Catnb remains undeleted.

View larger version (30K): [in a new window]   Fig. 8. ß-catenin is dispensable for maintenance and function of adult endocrine cells. Confocal immunofluorescence reveals that nearly all insulin+ islet cells are ß-catenin-deficient in PBKO adult pancreata (A,A'), whereas all amylase+ acinar cells retain ß-catenin expression (B,B'), just as in the developing organ. (C) Glucose tolerance tests of wild-type (n=5) and PBKO (n=5) adult mice. Points represent mean blood glucose for each timepoint following intraperitoneal glucose injection (2 mg/g body weight), error bars indicate standard deviation. There are no statistically significant differences between genotypes at any timepoint. is, islet.

View larger version (97K): [in a new window]   Fig. 9. ß-catenin deletion in adult exocrine cells. (A,B) X-gal staining (blue) on sections of adult R26R;Ela-CreERT double transgenic pancreata. In the absence of tamoxifen (TM) administration (A), a low level of TM-independent acinar labeling is observed (arrow), whereas widespread acinar labeling is observed two weeks after TM administration (B). No labeling is observed in ductal elements (arrowheads). (C-E') Confocal immunofluorescence to detect ß-catenin and amylase expression. All amylase+ cells in the TM-untreated pancreas retain ß-catenin expression (E), whereas many ß-catenin–/amylase+ cells can be found following TM treatment, including entire acini (arrowhead) comprising ß-catenin-deficient cells but exhibiting normal architecture (E').