Volume 143, Issue 2, Pages 481-492 (August 2012) Deficiency of Sbds in the Mouse Pancreas Leads to Features of Shwachman–Diamond Syndrome, With Loss of Zymogen Granules  Marina E. Tourlakis, Jian Zhong, Rikesh Gandhi, Siyi Zhang, Lingling Chen, Peter R. Durie, Johanna M. Rommens  Gastroenterology  Volume 143, Issue 2, Pages 481-492 (August 2012) DOI: 10.1053/j.gastro.2012.04.012 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Targeted loss of Sbds in the pancreas results in growth impairment. (A) Schematics of the missense (R126T) and floxed (CKO) alleles are shown with genotyping primers (see Supplementary Figure 1 for details) and representative PCR products. The CKO allele was generated by insertion of loxP sites in the first and second introns of Sbds. Residual Frt sites resulted from removal of a selection cassette that was used to generate the embryonic stem cell line. Excision of the CKO allele was achieved by crossing to a Ptf1a-Cre strain. Genotyping confirmed targeted excision (500-bp product, see Supplementary Figure 1 for details) in the pancreas. (B) Pancreata ablated for Sbds were only 0.31% of body mass as compared with 0.58% of control littermates at P30. Representative shown in (i) and compared in (ii). Growth impairment was specific to the pancreas (ii). SDS pancreas mouse models also displayed overall growth impairment with lower body mass and smaller size (P20, iii). Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Complete ablation of Sbds in the pancreas results in acinar cell hypoplasia as early as E18.5. Sections were stained with H&E. (A) Pancreas sections from control littermates and the disease-associated constitutive SDS model (SbdsR126T/R126T) appeared identical before birth (E18.5). (B) The fully ablated conditional model (SbdsP−/−) showed disrupted morphology at E18.5, and sparse amylase-positive cells (not shown), whereas the disease-associated conditional model (SbdsP−/R126T) resembled the constitutive model. Asterisks denote islets. Scale bars: 100 μm. Insets, higher magnification of the same section. Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 The SDS model pancreas shows SDS phenotypes. Histologic and immunohistochemical analysis of pancreata from control (SbdsP−/+, i) and SDS disease (SbdsP−/R126T, ii) models. (A) H&E (HE), (B) amylase, (C) insulin, (D) Dolichos biflorus agglutinin (DBA), (E) Oil red O (ORO). Asterisks denote islets. (A–C) P64, (D and E) P30. Scale bars: 100 μm. Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 The SDS model pancreas shows persistent acinar hypoplasia and dysplasia with decreased digestive enzyme levels. (A) Trichrome staining of littermate control (SbdsP−/+) and SDS disease model (SbdsP−/R126T) pancreata at 13 months highlighted the persistence of acinar cell hypoplasia (white arrowheads indicate residual acini), marked fatty infiltration, and residual connective tissue (stained blue). Asterisks denote islets. (B) At higher magnification, trichrome staining of control pancreas sections (P25) showed ordered acini stained dark red (i). SbdsP−/R126T littermate pancreas sections showed strikingly fainter staining (ii, arrowheads). Amylase immunostaining of a serial section indicated acinar cells that did not stain for amylase (iii, arrowheads) as well as discrete and sporadic amylase-positive cells. Mutant and control sections were stained using identical conditions. (C) Pancreatic transcript levels of α-amylase (Amy2a5) were significantly lower at P25 in disease models (n = 4) compared with controls (n = 3). Chymotrypsinogen (Ctrb1) levels were significantly lower in disease models at P15 (n = 4) and P25 (n = 4) compared with controls (n = 3 and 6, respectively). Scale bars: (A and B) 100 μm. Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Model pancreata have low levels of the 80S ribosomal complex and few zymogen granules. (A) Toluidine blue staining was notably weaker in mutants vs controls at 1 week of age (asterisks denote islets). (B) Representative ribosomal profiles from pancreas lysates (n ≥ 7 for all genotypes) showed reduced 80S ribosomal complexes. (C and D) Transmission electron microscopy revealed normal ultrastructure with reduced numbers of ZGs. ER, endoplasmic reticulum; M, mitochondrion; N, nucleus. (E) Glycoprotein 2 (Gp2) and syncollin (Sycn) messenger RNA levels were reduced significantly in mutant pancreata (black bars, n = 4) at P25 as compared with controls (gray bars, n = 6). Sycn messenger RNA levels were reduced significantly in mutant pancreata at P15 (black bars, n = 4) as compared with controls (gray bars, n = 3). Scale bars: (A) 100 μm, (C) 2 μm, and (D) 500 nm. Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 The endocrine compartment is impaired by Sbds ablation. (A) SbdsP−/R126T islets (asterisks) were immunoreactive for insulin and glucagon at P30. Scale bars: 100 μm. (Comparable results were observed with SbdsP−/− mice; not shown.) (B) Median islet diameter in the null model was smaller than controls at P30 (for details see Supplementary Table 4); however, serum insulin levels were comparable across all genotypes at this age (P = .53). (C) By 12 months, glucose tolerance tests indicated increased peak glucose levels (P = .018 and P = .023 at 15 and 30 minutes, respectively). Inset histograms show area under the curve, which was increased in the SbdsP−/− model (P = .031). A similar trend was suggested in the SbdsP−/R126T model but did not reach statistical significance when compared with littermate controls (area under the curve, P = .18). Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Longitudinal data highlight persistent growth restriction. (A) Average body mass of conditional models indicated reduced weight gain as compared with control littermates (data are shown for male mice). (B) Serum digestive enzyme levels were significantly lower in SbdsP−/R126T mice (n = 16) as compared with controls (n = 19) (see Supplementary Table 5 for details). (C) SbdsP−/R126T mice were refractory to diet manipulation (34% wt/wt fat), failing to gain weight or develop fat pads (see arrowheads) as compared with controls (data are shown for male mice). The high-fat chow is marked by blue-green color. Gastroenterology 2012 143, 481-492DOI: (10.1053/j.gastro.2012.04.012) Copyright © 2012 AGA Institute Terms and Conditions