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

Esni F, Täljedal IB, Perl AK, Cremer H, Christofori G, Semb H - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Umeå University, S-901 87 Umeå, Sweden.

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.

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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.
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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.

Mentions: To elucidate the molecular mechanisms behind the altered cell type segregation within islets of Langerhans in N-CAM-deficient mice, the subcellular distribution of molecules that are known to affect morphogenetic behaviors of cells or act as molecular markers for cell polarity were examined. Thus far, three members of the classic cadherins have been found to be expressed in islets, E-, N-, and R-cadherin (Begemann et al., 1990; Moller et al., 1992; Hutton et al., 1993; Dahl et al., 1996; Esni, F., and H. Semb, unpublished observations). Only N-cadherin and E-cadherin localize to cell–cell contacts (Begemann et al., 1990; Dahl et al., 1996), whereas R-cadherin distribute mainly in the cytoplasm (Dahl, U., F. Esni, and H. Semb, unpublished observations). Analysis of N-CAM-deficient mice revealed striking changes in the subcellular distribution of N-cadherin and E-cadherin, whereas R-cadherin was unaffected. As mentioned previously, these cadherins normally localize to all regions engaged in cell–cell adhesion without any apparent clustering (Begemann et al., 1990; Dahl et al., 1996; Fig. 4 a). In N-CAM-deficient mice, N-cadherin (Fig. 4, a–d) but also E-cadherin (data not shown) accumulate in discrete regions of cell–cell contacts, suggesting that N-CAM prevents clustering of cadherins within islets of Langerhans in control mice. Because cadherins are capable of affecting cortical F-actin organization and cell polarity, the subcellular distribution of F-actin and Na+/K+-ATPase (Hammerton et al., 1991; Drubin and Nelson, 1996), apical and basolateral epithelial cell polarity markers, respectively, were investigated in N-CAM-deficient mice. In control islets F-actin's preferential distribution is adjacent to cell–cell contacts (Fig. 4 e), whereas in N-CAM-deficient mice it accumulates together with the cadherins (Fig. 4, f–h). The visualization of the redistribution of N-cadherin, E-cadherin, and F-actin appears as multicellular rosette structures, which are composed of all endocrine cell types, and are reminiscent of the apical enrichment of E-cadherin and F-actin in exocrine acinar cells (insets in Fig. 4, d and h). We propose to refer to these structures as endocrine acini. The striking changes in the organization of cadherins and actin filaments in islets of N-CAM-deficient mice suggest that cell polarity may also be affected. In further support for alterations in islet cell polarity, the subcellular localization of the basolateral epithelial cell polarity marker Na+/K+-ATPase and the nuclei within islets of N-CAM-deficient is reminiscent of their distribution within fully polarized exocrine acinar cells (Fig. 5, compare a with c and d, and e with g and h). In normal control islets, Na+/ K+-ATPase colocalizes with N-cadherin and E-cadherin in cell contact regions (Fig. 5 b, and data not shown), whereas in islets of N-CAM-deficient mice Na+/K+-ATPase does not accumulate in regions with increased cadherin and F-actin clustering (Fig. 5, c and d). Furthermore, while islet cell nuclei are randomly distributed in control islets (Fig. 5 f), cell nuclei are preferentially localized to the basal regions of those islet cells that are organized in endocrine acini in N-CAM-deficient mice (Fig. 5, g and h). Similar to the effect on cell type segregation, changes in the subcellular localization of cadherins and F-actin in N-CAM-deficient mice were first observed at 4–5 wk of age. To examine whether transdifferentiation of endocrine cells into exocrine acinar cells could explain the appearance of the endocrine acinar structures, expression of acinar markers, such as amylase or carboxypeptidase, was investigated. However, none of these exocrine markers is expressed in islets of N-CAM +/− and N-CAM −/− mice, indicating that the appearance of acinar structures in islets of Langerhans does not resemble a transdifferentiation process (data not shown).


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

Esni F, Täljedal IB, Perl AK, Cremer H, Christofori G, Semb H - J. Cell Biol. (1999)

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.
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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.
Mentions: To elucidate the molecular mechanisms behind the altered cell type segregation within islets of Langerhans in N-CAM-deficient mice, the subcellular distribution of molecules that are known to affect morphogenetic behaviors of cells or act as molecular markers for cell polarity were examined. Thus far, three members of the classic cadherins have been found to be expressed in islets, E-, N-, and R-cadherin (Begemann et al., 1990; Moller et al., 1992; Hutton et al., 1993; Dahl et al., 1996; Esni, F., and H. Semb, unpublished observations). Only N-cadherin and E-cadherin localize to cell–cell contacts (Begemann et al., 1990; Dahl et al., 1996), whereas R-cadherin distribute mainly in the cytoplasm (Dahl, U., F. Esni, and H. Semb, unpublished observations). Analysis of N-CAM-deficient mice revealed striking changes in the subcellular distribution of N-cadherin and E-cadherin, whereas R-cadherin was unaffected. As mentioned previously, these cadherins normally localize to all regions engaged in cell–cell adhesion without any apparent clustering (Begemann et al., 1990; Dahl et al., 1996; Fig. 4 a). In N-CAM-deficient mice, N-cadherin (Fig. 4, a–d) but also E-cadherin (data not shown) accumulate in discrete regions of cell–cell contacts, suggesting that N-CAM prevents clustering of cadherins within islets of Langerhans in control mice. Because cadherins are capable of affecting cortical F-actin organization and cell polarity, the subcellular distribution of F-actin and Na+/K+-ATPase (Hammerton et al., 1991; Drubin and Nelson, 1996), apical and basolateral epithelial cell polarity markers, respectively, were investigated in N-CAM-deficient mice. In control islets F-actin's preferential distribution is adjacent to cell–cell contacts (Fig. 4 e), whereas in N-CAM-deficient mice it accumulates together with the cadherins (Fig. 4, f–h). The visualization of the redistribution of N-cadherin, E-cadherin, and F-actin appears as multicellular rosette structures, which are composed of all endocrine cell types, and are reminiscent of the apical enrichment of E-cadherin and F-actin in exocrine acinar cells (insets in Fig. 4, d and h). We propose to refer to these structures as endocrine acini. The striking changes in the organization of cadherins and actin filaments in islets of N-CAM-deficient mice suggest that cell polarity may also be affected. In further support for alterations in islet cell polarity, the subcellular localization of the basolateral epithelial cell polarity marker Na+/K+-ATPase and the nuclei within islets of N-CAM-deficient is reminiscent of their distribution within fully polarized exocrine acinar cells (Fig. 5, compare a with c and d, and e with g and h). In normal control islets, Na+/ K+-ATPase colocalizes with N-cadherin and E-cadherin in cell contact regions (Fig. 5 b, and data not shown), whereas in islets of N-CAM-deficient mice Na+/K+-ATPase does not accumulate in regions with increased cadherin and F-actin clustering (Fig. 5, c and d). Furthermore, while islet cell nuclei are randomly distributed in control islets (Fig. 5 f), cell nuclei are preferentially localized to the basal regions of those islet cells that are organized in endocrine acini in N-CAM-deficient mice (Fig. 5, g and h). Similar to the effect on cell type segregation, changes in the subcellular localization of cadherins and F-actin in N-CAM-deficient mice were first observed at 4–5 wk of age. To examine whether transdifferentiation of endocrine cells into exocrine acinar cells could explain the appearance of the endocrine acinar structures, expression of acinar markers, such as amylase or carboxypeptidase, was investigated. However, none of these exocrine markers is expressed in islets of N-CAM +/− and N-CAM −/− mice, indicating that the appearance of acinar structures in islets of Langerhans does not resemble a transdifferentiation process (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Umeå University, S-901 87 Umeå, Sweden.

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.

Show MeSH
Related in: MedlinePlus