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.

Liver and biliary system pathology in equine dysautonomia (grass sickness).

Hepatocellular and hepatobiliary damage was assessed in equine acute, subacute and chronic grass sickness cases (AGS, SAGS, CGS). Histopathological analysis showed that even in some early AGS cases enlarged hepatocytes, hepatocyte vacuolation indicative of lipid accumulation (steatosis), intrahepatocyte, canalicular and periportal deposition of pigments, frequent leucocyte infiltration and cholangitis occurred. Analysis of serum indicated significantly increased levels of unconjugated bilirubin in all groups and conjugated bilirubin in AGS and SAGS groups, increased levels of bile acids in some individuals from each group and significantly increased levels of glutamate dehydrogenase (GLDH) in AGS and SAGS cases. Conjugated bilirubin was significantly elevated in urine of AGS and SAGS cases. The evidence suggests that abnormal liver function involving moderate hepatocellular pathology in conjunction with steatosis and cholestasis may contribute to the pathogenesis of GS.

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.

Differential expression of cadherin-2 and cadherin-4 in the developing and adult zebrafish visual system.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. Although cadherins have been implicated in the development of the brain, including the visual system, in several vertebrate species, little is known of their role in zebrafish. In this study, we examined distribution of cadherin-2 (Cdh2, N-cadherin) in the visual system of developing and adult zebrafish using both immunocytochemical and in situ hybridization methods, and we compared Cdh2 distribution to that of the previously reported and closely related cadherin-4 (Cdh4, R-cadherin). As in other vertebrates, in zebrafish embryos Cdh2 was widely expressed in the early nervous system, but its expression became more restricted as development proceeded. Cdh4 was not detectable until later in development, at about the time when the first ganglion cells are generated. Cdh2 and Cdh4 were expressed in distinct regions of developing visual structures, including the lens. We hypothesize that the differential expression of these two cadherins in developing zebrafish visual structures reflects functionally different roles in the development of the vertebrate visual system.

Investigation of the susceptibility of equine autonomic neuronal cell lines, clonally derived from the same paravertebral ganglion, to toxic plasma from equine dysautonomia (grass sickness) cases.

In the autonomic nervous system (ANS) of equine grass sickness (GS) cases, some neurones show abnormal changes while neighbouring neurones are unaffected. To test whether noradrenergic neurones showed variable susceptibility to the GS toxin in culture, clonally-derived populations isolated from the same fetal thoracic sympathetic chain ganglion were challenged with plasma from GS cases previously shown to induce ANS damage when injected into normal horses. During the early stages of exposure to toxic plasma, cells within a clonal population showed variable susceptibility ranging from no obvious effect to characteristic patterns of pathology. However, after 3 days of exposure to toxic plasma all cells were killed. Dose response analysis on selected clonal populations showed no significant difference in TD(50) values.

Establishment and characterization of equine autonomic ganglion cell lines to enable direct testing of candidate toxins involved in equine dysautonomia (grass sickness).

To enable direct testing of a range of potential toxins or pathogens that might be involved in grass sickness, equine thoracic sympathetic chain ganglion cell lines were established from primary cell cultures by retroviral-mediated transduction of the temperature-sensitive mutant of the establishment oncogene encoding SV40 large T antigen. Morphological and behavioral features, temperature dependence, and immunocytochemical characteristics of the cell lines were investigated. The majority of cells were noradrenergic neurons in which dopamine-beta-hydroxylase, the enzyme that catalyzes norepinephrine synthesis, and neuropeptide Y coexisted. Cells treated with plasma from grass sickness cases that had previously been shown to induce autonomic nervous system damage when injected into normal horses showed significantly decreased mitochondrial function after 1 day. After 3 days exposure most cells showed severe degeneration in contrast to those treated with normal plasma. Liver and lung cell lines were also susceptible to plasma, suggesting that the toxin is not specifically neurotoxic.

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.