Identification and characterization of zebrafish ocular formation genes.

To study genes that are specifically expressed in the eyes, we employed microarray and in situ hybridization analyses to identify and characterize differentially expressed ocular genes in eyeless masterblind (mbl-/-) zebrafish (Danio rerio). Among 70 differentially expressed genes in the mbl-/- mutant identified by microarray analysis, 8 down-regulated genes were characterized, including 4 eye-specific genes, opsin 1 short-wave-sensitive 1 (opn1sw1), crystallinbetaa1b (cryba1b), crystallinbetaa2b (cryba2b), and crystallingamma M2d3 (crygm2d3); 2 eye and brain genes, ATPase, H+ transporting, lysosomal, V0 subunit c (atp6v0c) and basic leucine zipper and W2 domains 1a (bzw1a); and 2 constitutive genes, heat shock protein 8 (hspa8) and ribosomal protein L7a (rpl7a). In situ hybridization experiments confirmed down-regulation of these 8 ocular formation genes in mbl-/- zebrafish and showed their ocular and dynamic temporal expression patterns during zebrafish early development. Further, an automated literature analysis of the 70 differentially expressed genes identified a sub-network of genes with known associations, either with each other or with ocular structures or development, and shows how this study contributes to the current body of knowledge.     

Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

Background  

Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development.     

Expression profiling identifies novel Hh/Gli-regulated genes in developing zebrafish embryos

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.     

Identification and expression of soul/p22HBP genes in zebrafish

The SOUL/p22HBP family is an evolutionarily ancient group of heme binding proteins with a main function as cytosolic buffer against tetrapyrrole accumulation. Structural and biochemical evidence suggest specialized roles in blood formation, necrotic cell death and chemotaxis. To date, nothing is known about the precise activity and expression patterns of this class of heme binding proteins during development. The zebrafish genome possesses five soul genes belonging to two subgroups, and no p22HBP orthologous gene. Here, spatial and temporal expression patterns are reported for zebrafish soul1, soul2 and soul4 genes. All three soul genes are maternally transcribed, and their zygotic expression takes place in unique (heart, pharynx, yolk syncytial layer, brain, eyes, lateral line) and overlapping (pronephros, pituitary gland, olfactory and otic vesicle) regions of the zebrafish embryo. Our study constitutes the first detailed analysis of soul gene expression in metazoan development, and provides the basis to understand the genetics of tetrapyrrole metabolism in a wide range of embryonic processes.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

Identification and functional characterization of the CVOMTs and EOMTs genes promoters from Ocimum basilicum L.

Plant Cell, Tissue and Organ Culture (PCTOC) - Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced...     

Identification and characterization of endothelin converting activity in cultured bovine endothelial cells

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.     

Identification and expression analysis of kcnh2 genes in the zebrafish

Long QT syndrome is a disorder that is characterised by a prolonged QT-interval and can lead to fatal cardiac arrhythmias. Many animal models have been created to study congenital long QT syndrome. Of these, zebrafish models have involved targeting two different KCNH2 gene (long QT syndrome 2) orthologues, termed zerg-2 and zerg-3, with differing cardiac phenotypes. In order to clarify this situation, this study uses a bioinformatic approach to search the current zebrafish genome sequence (Zv7 and Zv8 builds) to investigate and locate all likely zebrafish orthologues of the human KCNH2 gene. Quantitative real-time RT-PCR was also used to determine the temporal and spatial gene expression profile of the zebrafish orthologues. The data support the conclusion that zerg-2 and zerg-3 are apparent orthologues of different human genes encoding potassium ion channels, but that their functions have switched compared to the respective human proteins.     

Identification and characterization of zebrafish semaphorin 6D

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.