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First published online 17 July 2008
doi: 10.1242/dev.013722

Development 135, 2757-2765 (2008)
Published by The Company of Biologists 2008
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Conditional ablation of Notch signaling in pancreatic development

Hassan Nakhai1,*, Jens T. Siveke1,*, Bettina Klein2, Lidia Mendoza-Torres1, Pawel K. Mazur1, Hana Algül1, Freddy Radtke3, Lothar Strobl4, Ursula Zimber-Strobl4 and Roland M. Schmid1,{dagger}

1 Department of Internal Medicine, Technical University of Munich, Ismaniger Strasse 22, 81675 Munich, Germany.
2 Institute of Immunology, Friedrich-Loeffler Institut, Paul-Ehrlich Strasse 28, 72076 Tuebingen, Germany.
3 ISREC, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
4 GSF-National Research Center for Environment and Health, Institute for Clinical Molecular Biology and Tumor Genetics, Marchioninistrasse 25, 81377 Munich, Germany.


Figure 1
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  		Fig. 1. Pancreas-specific Rbpj and Notch1/2KO mice. (A) Strategy for targeting the Rbpj locus to generate Rbpj+/f mice. The loxP sequences (arrowheads), exons (filled boxes), length of diagnostic restriction fragments and location of a 3'-probe (bar) used for Southern blotting are shown. EcoRI restriction enzyme sites (E) are indicated. (B) Southern blot analysis of Cre-mediated deletion in the following organs of an Ptf1a+/Cre(ex1);Rbpj+/- mouse: thymus (lane 1), spleen (2), liver (3), pancreas (4), kidney (5), head (6), lung (7), salivary gland (8), stomach (9), duodenum (10), and coecum (11). The positions and sizes of the fragments derived from the wild-type (WT), deleted, floxed and pseudogene alleles are indicated. (C) Newborn RbpjKO mice show increasing signs of growth retardation and die 4-5 days postpartum. (D-I) Macroscopic (D-F) and microscopic (G-I) X-gal staining analysis of intestinal tracts from newborn mice show X-gal+ pancreata (blue) from RbpjKO;R26R, Notch1/2KO;R26R and Rbpj+/-;R26R pups. Arrowhead in F indicates weakly branched ducts in pancreatic rudiment. neor, neomycin-resistance gene; HSV-tk, herpes simplex virus thymidine kinase gene; d, duodenum; l, liver; p, pancreas; sp, spleen; st, stomach. Scale bar: 100 µm.

 

Figure 2
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  		Fig. 2. Analysis of early pancreatic development in mutant embryos. (A-C) Pancreatic development of the respective mutant embryos at E13.5 as determined by PDX1 immunofluorescence (green). (E-G) Immunofluorescence for PDX1 (green) and HES1 (red) in pancreatic dorsal buds of mutant embryos at E12.5. (H-J) Double staining of pancreatic dorsal buds of mutant embryos for X-gal (blue) and glucagon (brown) at E11.5. (K-M) Nuclear expression of NGN3 in E13.5 mutant pancreata by immunohistochemistry. Arrowheads mark the areas in insets (enlarged 4x). Arrows in insets mark NGN3+ cells (black). (O-Q) Insulin expression in mutant pancreata at E13.5 by immunofluorescence (red, arrows). (S-U) Double-immunostaining for phospho-histone H3 (PHH3, black, arrows) and PDX1 (brown) at E13.5. (D,N,R,V) Quantification of the number of NGN3+, Insulin+ and PHH3+ cells, and size of area of PDX1+ cells, in buds of E13.5 Rbpj+/-;R26R, Notch1/2KO;R26R and RbpjKO;R26R embryos. Histograms show the mean size±s.d. for ventral and dorsal buds of three embryos each. Nuclei were counterstained with DAPI. Gluc, glucagon; vb, ventral bud; db, dorsal bud. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Early Cre-induced recombination of Notch1 and Notch2 alleles in the pancreatic epithelium. (A,B) Detection of early {alpha} cells in the dorsal bud by immunostaining for glucagon at E12.5. (A) Microdissection of pancreatic epithelial cells. (B) Note that glucagon+ cells (dark) are not dissected. (C,D) Schematic maps of the floxed Notch1 (C) and Notch2 (D) locus before and after Cre-induced recombination. The position and polarity of the primers used for amplification are represented by P1, P2 and P3 for Notch1, and P4, P5, P6 and P7 for Notch2 (red arrowheads). DNA was isolated from pancreatic epithelial cells of two Notch1/2KO embryos (lane 1 and lane 2). As a control, DNA from heterozygous Notch1/2+/f mice was used (lane 3).

 

Figure 4
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  		Fig. 4. Appearance of acinar cells in late stage of embryonic development of RbpjKO embryos. (A-F) Analysis of amylase expression at E14.5 (A-C) and E18.5 (D-F) in mutant pancreata. (G-I) Immunostaining of mutant pancreata with an antibody against the ductal marker CK19 (black, arrows) at day 3 postpartum (P3). (I) CK19 and X-gal (blue) co-staining of RbpjKO pancreas at P3. (J-L) Amylase staining (brown) of duct-like structures of RbpjKO pancreas at E18.5. In contrast to ventral bud (L), the most of dorsal duct-like structures are positive for amylase (J,K). (M-O) Amylase+ cells (green) from RbpjKO embryos express PDX1 (M, red) and PTF1A (O, red), detected by immunofluorescence, and are proliferating as determined by cytoplasmic amylase (brown) and nuclear BrdU (black) staining (N, arrow). Inset in M represents a 2.6x enlargement. (P) Double-immunofluorescence staining of acini from RbpjKO;R26R mice for amylase and β-galactosidase at P3. (Q) PCR analysis of DNA from microdissected acinar cells of RbpjKO (lane 1) and Rbpj+/- (lane 2) pancreata. Schematic maps of the floxed Rbpj locus before and after Cre recombination are shown. The position and polarity of the primers used for amplification are represented by P8 and P9 (red arrowheads). Nuclei were counterstained with DAPI. Amy, amylase; ac, acinar; m, mesentery. Scale bars: 50 µm (100 µm in D,E).

 

Figure 5
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  		Fig. 5. Endocrine development in RbpjKO, Notch1/2KO and Rbpj+/- pancreata. (A-L) Transverse sections from E18.5 Rbpj+/-, Notch1/2KO and RbpjKO;R26R embryos were analyzed for the expression of endocrine markers by immunohistochemistry. RbpjKO pancreas was stained by X-Gal (blue) in addition to the respective endocrine genes. l, liver. (M-O) X-gal staining of sections from Rbpj+/-;R26R, Notch1/2KO;R26R and RbpjKO;R26R embryos at E18.5. Arrows mark the areas in insets (enlarged 2x). (P-R) Double-immunofluorescence staining of islets with anti-glucagon (red) and anti-insulin antibodies (green). (S-U) The merged images show co-expression of glucagon and insulin with β-galactosidase in islets of knockouts and control pancreata. (V) Histogram representing the number of islets±s.d. in pancreata of three embryos for the indicated genotype. Ins, insulin; Gluc, glucagon; PP, pancreatic polypeptide. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2008