Figure 3

(A) Schematic representation of N-cadherin forms. N-cadherin contains a pro domain, 5 extracellular (EC1–EC5) domains, a transmembrane domain (62), and a cytosolic (CYTO) domain. Nonadhesive pro N-cadherin is proteolytically cleaved, creating mature N-cadherin (Mat). Additional processing generates signaling-competent N- and C- terminal fragments (NTF, CTF). (B) Schematic illustrates role of N-cadherin forms in chondrogenesis and synaptogenesis. Axons are shown in green and bungarotoxin-stained postsynaptic densities are red. (C) Representative N-cadherin Western blot reveals defects in processing in pmm2^m/m embryos. n = 3 experiments with 15 embryos per sample per experiment. (D) Quantification of individual protein forms. Error bars show SEM, Student’s t test, ***P < 0.01. (E and F) Confocal images of chondrocytes stained immunohistochemically with N-cadherin (red) or β-catenin (green). Cell surface is stained with WGA (blue). White arrows highlight N-cadherin location. In +/+ it is primarily found at the cell poles, but in m/m N-cadherin interactions persist on opposing cell membranes. The yellow inset is a 2.5× magnification of the original panels of N-cadherin on opposing membranes. Yellow arrows highlight β-catenin location, and white dotted line highlights N-cadherin located laterally in elongated cells. Scale bars: 10 μm. (G) Graphs quantitating N-cadherin and β-catenin localization. Data presented as percentage cells within an individual cartilage. n = 10 embryos per genotype per age over 3 experiments. Error bars show SEM, Student’s t test, ****P < 0.0001. (H) Schematic illustrates model of N-cadherin localization and processing during normal and disrupted chondrogenesis. (I) The level of cell surface N-cadherin present in +/+ and m/m embryos. Shown is the percentage of total cells that are GFP⁺, GFP⁺ and N-cadherin⁺, or N-cadherin⁺. n = 3 experiments of 15, with cells isolated from pools of 15 embryos per sample. Error bars show SEM.