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PubMedDate not supplied

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.

Animals↗

PubMed2001

Posttranscriptional silencing of cytochrome P4501A1 (CYP1A1) during zebrafish (Danio rerio) development.

Induction patterns of cytochrome P4501A1 (CYP1A1), an early biochemical marker of exposure to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) were investigated during zebrafish (Danio rerio) development. A zebrafish CYP1A1 cDNA fragment was cloned and used to detect CYP1A1 mRNA in embryos exposed to TCDD (1 or 10 nM). Induction of CYP1A1 activity was dependent on age and state of hatch. CYP1A1 mRNA was observed by 15 hr postfertilization. CYP1A1 protein and monooxygenase activity were not detected until 3 days postfertilization and after hatch, as determined by Western immunoblot analysis and ethoxyresorufin O-deethylase (EROD) activity, respectively. In contrast to embryos, concomitant induction of mRNA and activity was detected in juvenile zebrafish (3 days posthatch) after 6 hr of TCDD exposure. Asynchronous induction of CYP1A1 mRNA and activity during development may be a general regulatory mechanism, as similar ontogenetic expression of this gene was demonstrated in mouse embryos. To our knowledge, this is the first report of CYP1A1 posttranscriptional silencing during embryogenesis. Our data suggest that TCDD-mediated induction of CYP1A1 activity is regulated differentially in developing and mature systems.

Aging↗

PubMed1999

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.

Animals↗

PubMed2001

Cloning and developmental expression of a zebrafish meis2 homeobox gene.

We show here that a zebrafish meis2 gene homolog has a dynamic expression pattern in the developing mesoderm and central nervous system. Meis family homeodomain proteins are known to act as cofactors with other homeodomain proteins. We find expression of meis2.1 in the developing zebrafish hindbrain and somites, correlating with reported sites of zebrafish hox gene expression, as well as in presumptive cerebellum, midbrain, retina and ventral forebrain. The expression pattern shares some, but not all, features with that of murine Meis2.

Amino Acid Sequence↗

PubMed2006

The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo.

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.

Adenosine Triphosphatases↗

PubMed2006

Proteomic analysis of protein profiles during early development of the zebrafish, Danio rerio.

In the present study, profiles of protein expression were examined during early development of zebrafish, an increasingly popular experimental model in vertebrate development and human diseases. By 2-DE, an initial increase in protein spots from 6 h post-fertilization (hpf) to 8-10 hpf was observed. There was no dramatic change in protein profiles up to 18 hpf, but significant changes occurred in subsequent stages. Interestingly, 49% of the proteins detected at 6 hpf remained detectable by 1 week of age. To map the protein expression patterns in 2-D gels, MALDI-TOF/TOF MS was employed to identify selected protein spots from early embryos. 108 protein spots were found to match known proteins and they were derived from 55 distinct genes. Interestingly, 11 (20%) of them produced multiple protein isoforms or distinct cleavage products. Although deyolked embryos were used in the analysis, a large number of vitellogenin derivatives remained prominently present in the embryos. Other than these, most of the identified proteins are cytosolic, cytoskeletal and nuclear proteins, which are involved in diversified functions such as metabolism, cytoskeleton, translation, protein degradation, etc. Some of the proteins with interesting temporal expression profiles during development are further discussed.

Animals↗

PubMed2004

Hematopoietic gene expression profile in zebrafish kidney marrow.

The zebrafish kidney marrow is considered to be the organ of definitive hematopoiesis, analogous to the mammalian bone marrow. We have sequenced 26,143 ESTs and isolated 304 cDNAs with putative full-length ORF from a zebrafish kidney marrow cDNA library. The ESTs formed 7,742 assemblies, representing both previously identified zebrafish ESTs (56%) and recently discovered zebrafish ESTs (44%). About 30% of these EST assemblies have orthologues in humans, including 1,282 disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database. Comparison of the effective and regulatory molecules related to erythroid functions across species suggests a good conservation from zebrafish to human. Interestingly, both embryonic and adult zebrafish globin genes showed higher homology to the human embryonic globin genes than to the human fetal/adult ones, consistent with evo-devo correlation hypothesis. In addition, conservation of a whole set of transcription factors involved in globin gene switch suggests the regulatory network for such remodeling mechanism existed before the divergence of the teleost and the ancestor of mammals. We also carried out whole-mount mRNA in situ hybridization assays for 493 cDNAs and identified 80 genes (16%) with tissue-specific expression during the first five days of zebrafish development. Twenty-six of these genes were specifically expressed in hematopoietic or vascular tissues, including three previously unidentified zebrafish genes: coro1a, nephrosin, and dab2. Our results indicate that conserved genetic programs regulate vertebrate hematopoiesis and vasculogenesis, and support the role of the zebrafish as an important animal model for studying both normal development and the molecular pathogenesis of human blood diseases.

Animals↗

PubMed2006

lessen encodes a zebrafish trap100 required for enteric nervous system development.

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest. The developmental controls that govern the specification and patterning of the ENS are not well understood. To identify genes required for the formation of the vertebrate ENS, we preformed a genetic screen in zebrafish. We isolated the lessen (lsn) mutation that has a significant reduction in the number of ENS neurons as well as defects in other cranial neural crest derived structures. We show that the lsn gene encodes a zebrafish orthologue of Trap100, one of the subunits of the TRAP/mediator transcriptional regulation complex. A point mutation in trap100 causes a premature stop codon that truncates the protein, causing a loss of function. Antisense-mediated knockdown of trap100 causes an identical phenotype to lsn. During development trap100 is expressed in a dynamic tissue-specific expression pattern consistent with its function in ENS and jaw cartilage development. Analysis of neural crest markers revealed that the initial specification and migration of the neural crest is unaffected in lsn mutants. Phosphohistone H3 immunocytochemistry revealed that there is a significant reduction in proliferation of ENS precursors in lsn mutants. Using cell transplantation studies, we demonstrate that lsn/trap100 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest derived cartilages secondarily. Furthermore, we show that endoderm is essential for ENS development. These studies demonstrate that lsn/trap100 is not required for initial steps of cranial neural crest development and migration, but is essential for later proliferation of ENS precursors in the intestine.

Animals↗

PubMed2002

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.

Animals↗

PubMed2006

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.

Amino Acid Motifs↗

PubMed2004

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation.

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.

Animals↗

PubMed2006

Vertebrate MAX-1 is required for vascular patterning in zebrafish.

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.

Animals↗

PubMed2005

Neuronal calcium sensor-1 gene ncs-1a is essential for semicircular canal formation in zebrafish inner ear.

We have analyzed the functional role of neuronal calcium sensor-1 (Ncs-1) in zebrafish development. We identified two orthologs of the mammalian NCS-1 gene. Full-length cDNAs encoding zebrafish Ncs-1a and Ncs-1b polypeptides were cloned and characterized. Whole-mount in situ hybridization revealed that ncs-1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs-1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs-1a expression was detected primarily in the brain. Expression of ncs-1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs-1b was expressed at low levels throughout the brain. Knockdown of ncs-1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs-1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function.

Amino Acid Sequence↗

PubMed2005

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+)(CD33-)(CD38-)Rho(lo)(c-kit+) cells, enriched for hematopoietic stem/progenitor cells with (CD34+)(CD33-)(CD38-)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

ADP-ribosyl Cyclase 1↗

PubMed2004

Induction of reversible hemolytic anemia in living zebrafish using a novel small molecule.

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.

Anemia, Hemolytic↗

PubMed2003

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.

Amino Acid Sequence↗

PubMedDate not supplied

Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications.

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.

Animals↗

PubMed2006

The zebrafish cornea: structure and development.

PURPOSE: To evaluate the zebrafish as a model for the studies of corneal development and disease. METHODS: Zebrafish embryos and larvae at various stages of development were used for documenting corneal morphogenesis and differentiation. Corneal samples were collected from embryos, larvae, and adult zebrafish for histologic and electron microscopy analysis. Expression patterns of corneal polypeptides were investigated by immunostaining of sections. RESULTS: The zebrafish cornea develops rapidly during embryogenesis, so that its three major layers, the epithelium, the stroma, and the endothelium, are well formed by day 3 postfertilization. The subsequent steps of corneal differentiation, such as the thickening of the corneal stroma, proceed relatively slowly. Several polypeptides are highly enriched in the epithelium or the stroma of the larval and adult zebrafish cornea and are excellent markers of corneal differentiation. CONCLUSIONS: Development and differentiation of the zebrafish cornea are easily accessible to analysis. Anatomic and ultrastructural characterization of the zebrafish cornea demonstrates many similarities to the human cornea and provides the basis for the use of the zebrafish model both to analyze the basic genetic mechanisms of corneal development and to study the causes of corneal disease.

Animals↗