Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N‐cadherin, paraxial protocadherin, and VN‐cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E‐cadherin. Here, we report a partial cDNA with extensive sequence homology to R‐cadherin. Spatial and temporal expression of this putative zebrafish R‐cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R‐cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R‐cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R‐cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R‐cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1′‐dioctadecyl‐3,3,3′,3′, tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R‐cadherin message. In the adult, R‐cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R‐cadherin‐mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system. J. Comp. Neurol. 410:303–319, 1999. © 1999 Wiley‐Liss, Inc.

The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at ∼10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.

ABSTRACT:Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned. We investigated spatial and temporal expression of cdh1 during early embryogenesis. Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB). Developmental regulation of MHB formation involves wnt1 and pax2.1. wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure.