Cadherin-1, -2 and -4 expression in the cranial ganglia and lateral line system of developing zebrafish

Cadherins are cell adhesion molecules that play important roles in development of a variety of tissues and organs including the nervous system. In this study we analyzed expression patterns of three zebrafish classical (type I) cadherins (cadherin-1, -2, and -4) in the embryonic zebrafish cranial ganglia and lateral line system using in situ hybridization and immunohistochemical methods. All three cadherins exhibit distinct spatiotemporal patterns of expression during cranial ganglia and lateral line system development. cadherin-1 message was detected in the trigeminal and facial ganglia, in the lateral line ganglia, and in most of neuromasts in the lateral lines. Cadherin-2 mRNA and protein were expressed by the majority of the cranial ganglia and lateral line system. Both cadherins were found in embryos younger than 24 hours post fertilization as well as in 2-3-day old embryos and larvae. In contrast, cadherin-4 mRNA and protein expression was detected in embryos older than 30 hours post fertilization and limited to the trigeminal, statoacoustic, and vagal cranial ganglia, and the lateral line ganglia of older embryos and larvae.     

Protocadherin‐17 function in Zebrafish retinal development

Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the retina. Most studies have focused on examining functions of classic cadherins (e.g. N‐cadherin) in retinal development. There is little information on the function of protocadherins in the development of the vertebrate visual system. We previously showed that protocadherin‐17 mRNA was expressed in developing zebrafish retina during critical stages of the retinal development. To gain insight into protocadherin‐17 function in the formation of the retina, we analyzed eye development and differentiation of retinal cells in zebrafish embryos injected with protocadherin‐17 specific antisense morpholino oligonucleotides (MOs). Protocadherin‐17 knockdown embryos (pcdh17 morphants) had significantly reduced eyes due mainly to decreased cell proliferation. Differentiation of several retinal cell types (e.g. retinal ganglion cells) was also disrupted in the pcdh17 morphants. Phenotypic rescue was achieved by injection of protocadherin‐17 mRNA. Injection of a vivo‐protocadherin‐17 MO into one eye of embryonic zebrafish resulted in similar eye defects. Our results suggest that protocadherin‐17 plays an important role in the normal formation of the zebrafish retina. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H⁺-ATPase–dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.     

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naïve parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32–35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.     

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.     

Diversity of the cadherin family: evidence for eight new cadherins in nervous tissue.

To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation, Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation,Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

Hox gene expression reveals regionalization along the anteroposterior axis of the zebrafish notochord

 The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord’s role in imparting anteroposterior information to adjacent structures is less well understood, especially as the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Here we report that four zebrafish hox genes: hoxb1, hoxb5, hoxc6 and hoxc8 are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3’ in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date. Received: 30 March 1998 / Accepted: 16 June 1998     

Biochemical characterization and functional analysis of two type II classic cadherins, cadherin-6 and -14, and comparison with E-cadherin

Classic cadherins can be grouped based on their deduced primary structures. Among them the type I cadherins have been well characterized; however, little is known about non-type I cadherins. In this study we characterized two human type II cadherins, cadherin-6 and cadherin-14, using a cDNA transfection system. They were each detected as two bands electrophoretically, were expressed on the external cell surface at cell-cell contact sites, and were associated with caten- ins. Direct sequencing of the N-terminal amino acids showed that the two bands of cadherin-14 corresponded to precursor and mature forms, whereas the two bands of cadherin-6 both had the N-terminal sequence of the mature form. Unlike type I cadherins, both cadherin-6 and -14 were not protected from trypsin degradation by Ca2+. We evaluated their adhesive functions by a long term cell aggregation method. The results suggest that both cadherin-6 and -14 have cell-cell binding strengths virtually equivalent to that of E-cadherin and that their binding specificities are distinct from that of E-cadherin. Cadherin-6 and -14 interacted with each other in an incomplete manner. They have a QAI tripeptide in the first extracellular subdomain instead of the HAV motif that is characteristic of type I cadherins and is intimately involved in the adhesive function. The QAI tripeptide, however, appeared not to be involved in the adhesive functions of cadherin-6 and -14.     

Activity and distribution of paxillin,focal adhesion kinase,and cadherin indicate cooperative roles during zebrafish morphogenesis

We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.     

Regulation of nodal signalling and mesendoderm formation by TARAM-A, a TGFbeta-related type I receptor.

Nodal signalling is essential for many developmental events during vertebrate development, including the establishment of left-right asymmetry, of dorsoventral axis of the central nervous system, and endoderm and mesoderm formation. The zebrafish TGFbeta-related type I receptor, TARAM-A (Tar), is expressed in the prospective mesendodermal territory and, when activated, can transfate early blastomeres into endoderm, suggesting that Nodal and Tar may represent similar signalling pathways. We have analysed the functional relationships between those two pathways in zebrafish. We first demonstrate that tar and the zebrafish nodal genes cyc and sqt functionally interact. We also show that a dominant-negative isoform of Tar, TarMR, interferes specifically with the function of Cyc and Sqt in vitro, but does not interfere with the function of BMP2, another TGFbeta-related molecule. TarMR interferes also with Nodal signalling in vivo since it enhances the phenotype of embryos with weakened Nodal signalling. Overexpression of tarMR in wild-type embryos interfered with the formation of endoderm-derived structures. Conversely, overexpression of tar enlarged the presumptive mesendodermal region at the onset of gastrulation. Together, our results point to Tar as an essential factor for endoderm formation and an important modulator of Nodal signalling, potentially representing one of the Nodal receptors. (c)2001 Elsevier Science.     

Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.     

Roles for Zebrafish Focal Adhesion Kinase in Notochord and Somite Morphogenesis

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.