Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development

Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced.     

Mammalian protein homologous to VAT-1 of Torpedo californica: Isolation from Ehrlich ascites tumor cells,biochemical characterization,and organization of its gene

Recently, interest has focused on the human gene encoding the putative protein homologous to VAT-1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT-1 protein of Torpedo californica. This VAT-1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT-1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT-1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT-1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT-1 homolog with an extended exon 1 is proposed. VAT-1 and the mammalian VAT-1 homolog form a subgroup within the protein superfamily of medium-chain dehydrogenases/reductases. J. Cell. Biochem. 69:304–315, 1998. © 1998 Wiley-Liss, Inc.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.     

Developmental expression of zebrafish emx1 during early embryogenesis

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. Here we report a zebrafish emx family gene, emx1, which is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; we propose to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3.     

The isolation, characterization, and expression of a novel GDF11 gene and a second myostatin form in zebrafish, Danio rerio

In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-beta superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-beta superfamily array exists in primitive vertebrates than previously thought.     

Characterization and expression of a presomitic mesoderm-specific<Emphasis Type="Italic"> mespo</Emphasis> gene in zebrafish

A complete zebrafish mespo cDNA encoding a protein of 131 amino acids with a bHLH domain in the C-terminal has been isolated. The bHLH domain of zebrafish Mespo is highly similar to those in the mouse, chick and Xenopus, sharing 82.4%, 80.4% and 74.5% amino acid identity, respectively. At 50% epiboly, the zebrafish mespo is first detected in the marginal zone of the blastoderm but excluding the prospective shield. Subsequently, mespo expression is intensified in the involuting mesoderm at 60% epiboly, and then restricted to the presomitic mesoderm (PSM) at 95% epiboly. At the 1-somite stage, mespo expression becomes reduced in the most rostral PSM. During segmentation, mespo expression is gradually downregulated at the most rostral segmental plate where cells are being coalesced to form somites. In spadetail mutant embryos, most of mespo-expressing cells were missing.     

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.     

Zebrafish cypher is important for somite formation and heart development

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.     

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.     

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.     

Expression pattern of Siaz gene during the zebrafish embryonic development

Siah is a mammalian homolog of Drosophila seven in absentia (SINA). Here we report the identification of a new member of the SINA/Siah gene family. This new gene, designated Siaz, was found in zebrafish, and its product is predicted to share extensive amino acid sequence homology with Drosophila SINA. Siaz is maternally inherited, with zygotic expression in all blastomeres starting at the mid-blastula transition. After the 20-somite stage, Siaz expression occurs in a stage-specific manner in particular regions, including the brain, eye, cranial cavity, otic vesicle, optic chiasm and gut.     

Expression of mt2 and smt-B upon cadmium exposure and cold shock in zebrafish (Danio rerio)

Metallothionein-2 (mt2) and similar to metallothionein-B (smt-B) are included in the MT gene family. The objective of this study was to compare mt2 and smt-B messenger (m)RNA expressions after cadmium exposure and cold shock with whole-mount in situ hybridization in immature zebrafish (Danio rerio) and with a semi-quantitative RT-PCR in mature zebrafish. Three-day post-fertilization (dpf) larvae were treated with 0, 0.08, 0.26, and 0.89 microM cadmium for 24 and 48 h, and some larvae were challenged with a normal (28.5 degrees C) or low temperature (12 degrees C) for 12, 24, and 48 h. Results were obtained. (1) During embryonic and larval development, mt2 mRNA existed at 6 h post-fertilization (hpf), and the level rapidly increased to 24 hpf, then it gradually increased with further larval growth. smt-B was found at 12 hpf, and it also rapidly increased to 24 hpf, but remained constant during further larval development. (2) The mt2 mRNA signals and whole-body Cd contents displayed dose- and time-dependent responses after Cd exposure. After cold shock, mt2 mRNA signals also showed time-dependent expression. But smt-B mRNA signals were not appeared by either challenge. Besides, mature zebrafish were treated with 1.78 microM Cd and found that the highest levels of smt-B mRNA (smt-B/beta-actin) appeared in brain, and seems a reverse expression between smt-B mRNA and mt2 in brain after Cd exposure. Apparently, mt2 is possibly more relevant to Cd detoxification and cold shock adaptation in zebrafish larvae compared to smt-B, but smt-B might be related to certain physiological functions in neural (or brain) of mature zebrafish.