N-cadherin is dispensable for pancreas development but required for beta-cell granule turnover

Abstract

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N-cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N-cadherin during pancreas formation and function we generated a tissue-specific knockout of N-cadherin in the early pancreatic epithelium by inter-crossing N-cadherin-floxed mice with Pdx1Cre mice. Analysis of pancreas-specific ablation of N-cadherin demonstrates that N-cadherin is dispensable for pancreatic development, but required for beta-cell granule turnover. The number of insulin secretory granules is significantly reduced in N-cadherin-deficient beta-cells, and as a consequence insulin secretion is decreased.

Figure 1. N-cadherin expression in embryonic pancreas

N-cadherin is coexpressed with Pdx1 in the pancreatic epithelium E10.5-13.5 (a). At E15.5, N-cadherin is expressed in a subset of Sox9+ ductal cells, hormone producing cells (insulin⁺ and glucagon ⁺ cells), but not in amylase⁺ cells (b). The inset in N-cadherin Sox9 staining is from a different picture. Staining in green (middle) is indicated with a dotted line in red (left). E; embryonic day. Bars 20 μm, inset 10 μm.

Figure 2. N-cadherin expression in the conditional N-cadherin knockout mouse

N-cadherin is expressed throughout the wt epithelium at E13.5. The recombination efficiency varies from 5% to 95% in cKO embryos (a). At E15.5, approximately 50% of the cells express N-cadherin, while the recombination is complete at E18.5 and in adults (b). Cells with asterisk indicate N-cadherin-ablated cells. R26RLacZ were bred with Pdx1Cre to detect recombination efficiency. Analysis of R26RLacZ/Pdx1Cre mice showed that the recombination efficiency varied at E13.5, while all cells expressed βGal from E15.5 (c). Western blot analysis shows a complete deletion of N-cadherin in adult islets (d). Staining in green (right) is indicated with a dotted line in red (left). Tuba1a; α-Tubulin. Bars 20 μm.

Figure 3. N-cadherin is dispensable for pancreas morphology

No morphology changes between wt and cKO were observed (a). Sections throughout E15.5 pancreases were stained with Ins and Ecad, or Glu, Som, PP and Ecad, or Amy and Ecad. The areas of the different markers were measured and compared to the Ecad area. No differences were observed (b). Insulin was measured in adults by ELISA and no differences were observed. We also counted Glu⁺ cells versus Ins⁺ cells, PP⁺ cells versus Ins⁺ cells, and Som⁺ cells versus Ins⁺ cells. The ratios between different hormones were normal (c). Bars 20 μm.

Figure 4. N-cadherin is important for β-cell granule turnover and secretion

The amounts of mature granules were significantly lower in the cKO compared to wt (P<0.0001) (a,b). In addition, 16% of all mutant β-cells show a dramatic reduction in granule numbers (asterisk) (a). Even if the fraction of β-cells with very few insulin granules was not included in the data, the difference between wt and cKO was statistical significant (P<0.0001) (c). There is no significant difference in the number of immature granules (d). Significant value is indicated as ***p <0.001 and determined by Student's t-test (n=3). Stimulation with low glucose (2.8mM) results in a significant decrease compared to wt (p-value 0.0253). Similar observation was seen at high glucose (16.7mM), however this was not statistical significant (p-value 0.2217) (e). Significant value is indicated as *p <0.05 and determined by Student's t-test (n=7). Error bars represent standard error from the mean (±SEM). Bars 2 μm.