Cell Proliferation and Collective Cell Migration During Zebrafish Lateral Line System Development Are Regulated by Ncam/Fgf-Receptor Interactions.

The posterior lateral line system (pLLS) of aquatic animals comprises small clustered mechanosensory organs along the side of the animal. They develop from proneuromasts, which are deposited from a migratory primordium on its way to the tip of the tail. We here show, that the Neural Cell Adhesion Molecule Ncam1b is an integral part of the pathways initiating and regulating the development of the pLLS in zebrafish. We find that morpholino-knockdowns of ncam1b (i) reduce cell proliferation within the primordium, (ii) reduce the expression of Fgf target gene erm, (iii) severely affect proneuromast formation, and (iv) affect primordium migration. Ncam1b directly interacts with Fgf receptor Fgfr1a, and a knockdown of fgfr1a causes similar phenotypic changes as observed in ncam1b-morphants. We conclude that Ncam1b is involved in activating proliferation by triggering the expression of erm. In addition, we demonstrate that Ncam1b is required for the expression of chemokine receptor Cxcr7b, which is crucial for directed primordial migration. Finally, we show that the knockdown of ncam1b destabilizes proneuromasts, suggesting a further function of Ncam1b in strengthening the cohesion of proneuromast cells.

Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function.

Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.

Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio.

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

Ncam1a and Ncam1b: two carriers of polysialic acid with different functions in the developing zebrafish nervous system.

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.

Molecular evolution and expression of zebrafish St8SiaIII, an alpha-2,8-sialyltransferase involved in myotome development.

Enzymes of the St8Sia family, a subgroup of the glycosyltransferases, mediate the transfer of sialic acid to glycoproteins or glycolipids. Here, we describe the cloning of the zebrafish St8SiaIII gene and study its developmental activity. A conserved synteny relationship among vertebrate chromosome regions containing St8SiaIII loci underscores an ancient duplication of this gene in the teleost fish lineage and a specific secondary loss of one paralog in the zebrafish. The single zebrafish St8SiaIII enzyme, which is expected to function as an oligosialyltransferase, lacks maternal activity, is weakly expressed during nervous system development, and shows a highly dynamic expression pattern in somites and somite-derived structures. Morpholino knock-down of St8SiaIII leads to anomalous somite morphologies, including defects in segment boundary formation and myotendious-junction integrity. These phenotypes hint for a basic activity of zebrafish St8SiaIII during segmentation and somite formation, providing novel evidence for a non-neuronal function of sialyltransferases during vertebrate development.