Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets

Abstract

Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells have established that cellular cohesiveness, mediated by cell adhesion molecules, is important in determining the organization of cells within tissue and organs. We have employed N-CAM-deficient mice to determine whether N-CAM plays a functional role in the proper segregation of cells during the development of islets of Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing alpha cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected reduction of cell-cell adhesion in N-CAM-deficient mice, a significant increase in the clustering of cadherins, F-actin, and cell-cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei and Na+/K+-ATPase indicate that islet cell polarity is also affected. Finally, degranulation of beta cells suggests that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type segregation during organogenesis. Possible mechanisms underlying this phenomenon may include changes in cadherin-mediated adhesion and cell polarity.

Figure 2

Histological analysis of adult wild-type and N-CAM-deficient pancreata. (a–d) Hematoxylin-eosin stainings of paraffin sections of control (a and c, +/+) and N-CAM −/− (b and d, −/−) mice. Based on hematoxylin-eosin stainings, pancreata of N-CAM-deficient mice appear normal (data not shown for N-CAM +/− pancreata). (e-g) Immunofluorescence stainings of sections from adult wild-type (e, +/+), N-CAM +/− (f, +/−), and N-CAM −/− (g, −/−) pancreata with anti-N-CAM polyclonal antibodies. (h) Immunoblotting analysis of pancreatic islet extracts from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) animals, using anti-N-CAM polyclonal antibodies. While N-CAM is completely absent in N-CAM −/− islets, N-CAM +/− islets express ∼50% of wild-type islets. Bars: (a, b, and e–g) 20 μm; (c and d) 10 μm.

Figure 1

N-CAM expression in developing and adult pancreas. (a and b) Immunoperoxidase detection of N-CAM (a) and PDX1 (b), a marker for pancreatic endoderm, on consecutive transversal sections of 9.5 dpc pancreas. N-CAM is expressed in the pancreatic endoderm and in the mesenchyme, which surrounds both the ventral (vb) and dorsal (db) pancreatic buds. Each pancreatic bud is indicated by a broken line. (c and d) Double immunofluorescence staining of a transversal section of an 11.5 dpc pancreas with polyclonal antibodies against N-CAM (c) and glucagon (d). N-CAM is expressed in mesenchymal cells (m) and in clusters of α cells (arrows), but not in primitive epithelial cells (ep) which are indicated by a broken line. (e and f) Double immunofluorescence staining of 17.5 dpc pancreas with polyclonal antibodies against N-CAM (e) and insulin (f). At this stage N-CAM is selectively expressed in islet-like clusters of endocrine cells. Exocrine tissue is indicated by e. (g) Immunofluorescence staining of adult pancreas with polyclonal antibodies against N-CAM, showing specific expression in pancreatic islets. Exocrine tissue is indicated by e. (h) Immunoblotting analysis of extracts from pancreatic islets (C57) and the insulinoma cell line βTC4 (βTC; Efrat et al., 1988). The major N-CAM isoform expressed in islets is N-CAM-120. Bars, 20 μm.

Figure 3

Distribution of glucagon-producing α cells in pancreatic islets of control and N-CAM-deficient mice. Immunofluorescence staining of adult control (a, +/+), N-CAM +/− (b, +/−), and N-CAM −/− (c, −/−) mice with polyclonal antibodies against glucagon. In control animals the majority of α cells are localized in the periphery of islets. However, in N-CAM +/− and N-CAM −/− mice the α cells become more randomly distributed within the islets, referred to as mixed islets in Table I. Bar, 20 μm.

Figure 4

Subcellular organization of N-cadherin and F-actin is altered in pancreatic islets of N-CAM mutant mice. (a–d) Immunofluorescence stainings of sections from adult control (a, +/+), N-CAM +/− (b, +/−), and N-CAM −/− (c and d, −/−) pancreata with anti-N-cadherin mAb. Inset in d shows E-cadherin distribution in an exocrine acinus. (e–h) Staining of F-actin on sections from adult control (e, +/+), N-CAM +/− (f, +/−), and N-CAM −/− (g and h, −/−) pancreata with rhodamine-phalloidin. Inset in h shows F-actin distribution in an exocrine acinus. Arrows indicate rosette-like structures, or endocrine acini. The subcellular localization of N-cadherin and F-actin is reminiscent of the distribution of E-cadherin and F-actin in exocrine acini. Bars, 10 μm.

Figure 5

Subcellular localization of cell polarity markers suggests altered cell polarity in pancreatic islets of N-CAM-deficient mice. (a) Double immunofluorescence staining of an exocrine acinus in section of adult control (+/+) pancreas with anti-Na⁺/K⁺-ATPase polyclonal antibody (FITC) and anti-E-cadherin mAb (Cy3). While Na⁺/K⁺-ATPase colocalizes with E-cadherin in lateral cell–cell contacts, the molecule does not accumulate together with E-cad-herin in adherens junctions. (b–d) Double immunofluorescence stainings of islet cells in sections from adult control (b, +/+), N-CAM +/− (c, +/−), and N-CAM −/− (d, −/−) pancreata with anti-Na⁺/K⁺-ATPase polyclonal antibody (FITC) and anti-N-cadherin mAb (Cy3). Normally Na⁺/K⁺-ATPase colocalizes with N-cadherin in most islet cell–cell contacts (b). However, in N-CAM-defi-cient mice Na⁺/K⁺-ATPase does not accumulate together with N-cadherin in the apical regions of endocrine acini (c and d). (e) Exocrine acinus in section of adult control (+/+) pancreas immunostained with anti-E-cadherin mAb. Nuclei were stained with DAPI. E-cadherin is clustered in the apical region of lateral cell–cell contacts, while nuclei are preferentially distributed in the basal region of the cells. (f–h) Islet cells in sections of adult control (f, +/+), N-CAM +/− (g, +/−), and N-CAM −/− (h, −/−) pancreata immunostained with anti-N-cadherin mAb. Nuclei were stained with DAPI. In contrast to in control mice, the redistributions of N-cadherin and nuclei in endocrine acini of N-CAM +/− (g) and N-CAM −/− (h) mice are reminiscent of the localization of E-cadherin and nuclei in exocrine acini (e). Bar, 10 μm.

Figure 6

N-CAM-deficient mice exhibit ultrastructural alterations in islet cells. Electron photomicrographs of islets from control (a, +/+) and homozygous (b–d, −/−) animals. Because the ultrastructural changes were the same in heterozygous and homozygous mutants, only the data from the homozygous mice is shown. In b, and at higher magnification in c, clustering of cell–cell junctions, including desmosomes (arrowheads in c) and adherens type junctions (brackets in c), between four β cells are shown. In b three β cells contain a diminished number of secretory granules. Arrows in b-d indicate accumulation of residual bodies. In d residual bodies contain secretory granules. Dilation of rough endoplasmic reticulum was observed in β cells (asterisks in c) and α cells (data not shown). Bars: (a) 1 μm; (b) 2 μm; (c and d) 0.5 μm.

Figure 7

N-CAM affects the epithelial cell morphology of cadherin-expressing L cells. Parental L cells and L cells transfected with cDNAs for murine E-cadherin (LE; Nose et al., 1988) and N-cadherin (LN; Miyatani et al., 1989) were transiently transfected with cDNAs encoding murine N-CAM-120, N-CAM-140, and N-CAM-180. Except for N-CAM-120, which did not localize to the cell surface, all N-CAM isoforms exhibited similar effects on L cell morphology, independent of cadherin expression. Results from parental L cells and LE cells transfected with N-CAM-140 are shown. (a and b) Phase-contrast images of L cells (a) and LE cells (b). (c and d) Fluorescence of eGFP in L cells (c) and LE cells (d) transfected with eGFP alone. No change in cell morphology was observed. (e and f) Immunofluorescence staining of L cells (e) and LE cells (f) transfected with N-CAM-140. Expression of N-CAM conferred similar effects on cell morphology independently of whether the transfected cells exhibited a fibroblast-like or epithelial-like cellular phenotype. N-CAM induced extensive neurite-like extensions or filopodia on the cells, regardless of the mesenchymal or epithelial phenotype of the cell lines. In LE cells the majority of the transfected cells left the monolayer and migrated on top of      neighboring cells. (g–j) Double immunofluorescence stainings of N-CAM-140 transfected LE cells with anti-N-CAM polyclonal antibody (g and i) and anti-E-cadherin mAb (h and j). The majority of the transfected cells lost their typical epithelial phenotype upon N-CAM expression (g and h), however, occasionally, transfected cells remained within the monolayer (i and j). While the polar distribution of E-cadherin was lost in the former cells, it remained unchanged in the latter cells. Bars, 20 μm.