Cell adhesion molecule cadherin-6 function in zebrafish cranial and lateral line ganglia development.

Cadherins regulate the vertebrate nervous system development. We previously showed that cadherin-6 message (cdh6) was strongly expressed in the majority of the embryonic zebrafish cranial and lateral line ganglia during their development. Here, we present evidence that cdh6 has specific functions during cranial and lateral line ganglia and nerve development. We analyzed the consequences of cdh6 loss-of-function on cranial ganglion and nerve differentiation in zebrafish embryos. Embryos injected with zebrafish cdh6 specific antisense morpholino oligonucleotides (MOs, which suppress gene expression during development; cdh6 morphant embryos) displayed a specific phenotype, including (i) altered shape and reduced development of a subset of the cranial and lateral line ganglia (e.g., the statoacoustic ganglion and vagal ganglion) and (ii) cranial nerves were abnormally formed. These data illustrate an important role for cdh6 in the formation of cranial ganglia and their nerves.

Cloning and expression analysis of cadherin7 in the central nervous system of the embryonic zebrafish.

Cadherin cell adhesion molecules exhibit unique expression patterns during development of the vertebrate central nervous system. In this study, we obtained a full-length cDNA of a novel zebrafish cadherin using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of this molecule is most similar to the published amino acid sequences of chicken and mammalian cadherin7 (Cdh7), a member of the type II cadherin subfamily. cadherin7 message (cdh7) expression in embryonic zebrafish was studied using in situ hybridization and RT-PCR methods. cdh7 expression begins at about 12h postfertilization (hpf) in a small patch in the anterior neural keel, and along the midline of the posterior neural keel. By 24 hpf, cdh7 expression in the brain shows a distinct segmental pattern that reflects the neuromeric organization of the brain, while its expression domain in the spinal cord is continuous, but confined to the middle region of the spinal cord. As development proceeds, cdh7 expression is detected in more regions of the brain, including the major visual structures in the fore- and midbrains, while its expression domain in the hindbrain becomes more restricted, and its expression in the spinal cord becomes undetectable. cdh7 expression becomes reduced in 3-day old embryos. Our results show that cdh7 expression in the zebrafish developing central nervous system is both spatially and temporally regulated.

Differential expression of photoreceptor-specific genes in the retina of a zebrafish cadherin2 mutant glass onion and zebrafish cadherin4 morphants.

Cadherins are Ca2+ -dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g. rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.

Cadherin-2 function in the cranial ganglia and lateral line system of developing zebrafish.

Cadherins are cell surface molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin-2 (also called N-cadherin), a member of classic cadherin subfamily, has been shown to play important roles in development of a variety of tissues and organs, including the nervous system. We recently reported that cadherin-2 was strongly expressed by the majority of cranial ganglia and lateral line system of developing zebrafish. To gain insight into cadherin-2 role in the formation of these structures, we have used several markers to analyze zebrafish embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO). We find that development of several cranial ganglia, including the trigeminal, facial, and vagal ganglia, and the lateral line ganglia and neuromasts of the cdh2MO-injected embryos are severely disrupted. These phenotypes were confirmed by analyzing a cadherin-2 mutant, glass onion. Our results suggest that cadherin-2 function is crucial for the normal formation of the zebrafish lateral line system and a subset of cranial ganglia.

Cadherin-1, -2, and -11 expression and cadherin-2 function in the pectoral limb bud and fin of the developing zebrafish.

Cadherins are cell adhesion molecules that play important roles in development of a variety of organs, including the vertebrate limb. In this study, we analyze cadherin expression patterns in the embryonic zebrafish pectoral limb buds and larval pectoral fins by using both in situ hybridization and immunocytochemical methods. cadherin-1 is detected in the epidermis of the embryonic limb buds and the larval pectoral fins. Cadherin-2 is expressed in the pectoral limb bud mesenchyme and chondrogenic condensation. As development proceeds, cadherin-2 expression is detected in newly differentiated pectoral fin endoskeleton, but its expression is greatly down-regulated in the fin endoskeleton of larval zebrafish. cadherin-11 is found in the basal region of the embryonic limb buds and in the proximal endoskeleton of the larval pectoral fins. Interfering with cadherin-2 function using two specific antisense morpholino oligonucleotides disrupts formation of the chondrogenic condensation/endoskeleton, suggesting that cadherin-2 is crucial for the normal development of the zebrafish pectoral fins.

Cadherin expression in the inner ear of developing zebrafish.

Cadherins are cell adhesion molecules that have been implicated in development of a variety of organs including the ear. In this study we analyzed expression patterns of three zebrafish cadherins (Cadherin-2, -4, and -11) in the embryonic and larval zebrafish inner ear using both in situ hybridization and immunocytochemical methods. All three Cadherins exhibit distinct spatiotemporal patterns of expression during otic vesicle morphogenesis. Cadherin-2 and Cadherin-4 proteins and their respective mRNAs were detected mainly in the sensory patches and the statoacoustic ganglion (SAg), respectively. In contrast, cadherin-11mRNA was widely expressed earlier in the otic placode, and later became restricted to a subset of cells in the inner ear, including hair cells.

Up-regulation of cadherin-2 and cadherin-4 in regenerating visual structures of adult zebrafish.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. In this study, we examined expression of zebrafish cadherin-2 (Cdh2, N-cadherin) and cadherin-4 (Cdh4, R-cadherin) in the visual system of adult zebrafish after eye or optic nerve lesions using immunocytochemistry and immunoblotting. Both Cdh2 and Cdh4 immunoreactivities were specifically up-regulated in regenerating retina and/or the optic pathway. Furthermore, temporal expression patterns of these two cadherins were distinct during the regeneration of the injured tissues. Cadherins have been shown to regulate axonal outgrowth in the developing nervous system, but this is the first report, to our knowledge, of increased cadherin expression associated with axonal regeneration in the vertebrate central nervous system. Our results suggest that both Cdh2 and Cdh4 may be important for regeneration of injured retinal ganglion cell axons.

Cadherin-4 expression in the zebrafish central nervous system and regulation by ventral midline signaling.

In this study we show that zebrafish cadherin-4 (R-cadherin) transcript (cad4) and protein are expressed in several defined regions in the embryonic forebrain and in distinctive clusters in the hindbrain and spinal cord. This is the first report of a segmental pattern of expression of cadherin-4 in the hindbrain and spinal cord. Expression domains of cadherin-4 transcript and protein were compared with that of pax6.1. In the forebrain of zebrafish embryos, cad4 and pax6.1 expression domains overlapped extensively, although they were not completely coincident. Injection of pax6.1 mRNA resulted in an increase in cad4 expression, whereas overexpression of sonic hedgehog (shh), a midline signaling molecule that reduces pax6.1 expression, caused a reduction in cad4 expression throughout the brain. cad4 expression was increased in both forebrain and hindbrain in cyclops mutant embryos, which have a defect in midline signaling and an enlarged expression domain of pax6.1. These results suggest that zebrafish cadherin-4 may play a role in organization of neuronal architecture throughout the neural axis, and that its expression is regulated by a ventral midline signaling pathway that involves shh and pax6.1.

Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1.

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development.

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.

Mutant cadherin affects epithelial morphogenesis and invasion, but not transformation.

MDCK cells were engineered to reversibly express mutant E-cadherin protein with a large extracellular deletion. Mutant cadherin overexpression reduced the expression of endogenous E- and K-cadherins in MDCK cells to negligible levels, resulting in decreased cell adhesion. Despite severe impairment of the cadherin adhesion system, cells overexpressing mutant E-cadherin formed fluid-filled cysts in collagen gel cultures and responded to hepatocyte growth factor/scatter factor (HGF/SF) that induced cellular extension formation with a frequency similar to that of control cysts. However, cells were shed from cyst walls into the lumen and into the collagen matrix prior to and during HGF/SF induced tubule extension. Despite the propensity for cell dissociation, MDCK cells lacking cadherin adhesion molecules were not capable of anchorage-independent growth in soft agar and cell proliferation rate was not affected. Thus, cadherin loss does not induce transformation, despite inducing an invasive phenotype, a later stage of tumor progression. These experiments are especially relevant to tumor progression in cells with altered E-cadherin expression, particularly tumor samples with identified E-cadherin extracellular domain genomic mutations.

Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly.

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

Spatial correspondence between R-cadherin expression domains and retinal ganglion cell axons in developing zebrafish.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R-cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R-cadherin in situ hybridization to explicitly examine the association ofretinal axons and brain regions expressing R-cadherin message. We found that in zebrafish embryos at 46-54 hours postfertilization, DiI-labeled retinal axons were closely associated with cells expressing R-cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R-cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons.

R-cadherin expression in the developing and adult zebrafish visual system.

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.

Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression.

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 approximately 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.

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion.

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.

Cadherin cell adhesion molecules in differentiation and embryogenesis.

The cadherin gene superfamily of calcium-dependent cell-cell adhesion molecules contains more than 40 members. We summarize functions attributed to these proteins, especially their roles in cellular differentiation and embryogenesis. We also describe hierarchies of protein-protein interactions between cadherins and cadherin-associated proteins (catenins). Several signal transduction pathways converge on, and diverge from, the cadherin/catenin complex to regulate its function; we speculate on roles of these signaling processes for cell structure and function. This review provides a framework for interpretation of developmental functions of cadherin cell adhesion molecules.

Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.

The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin. In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin. Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules.

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.