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PubMed2002

Pigment pattern formation in zebrafish: a model for developmental genetics and the evolution of form.

The zebrafish Danio rerio is an emerging model organism for understanding vertebrate development and genetics. One trait of both historical and recent interest is the pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. In zebrafish, an embryonic and early larval pigment pattern consists of several stripes of melanophores and iridophores, whereas xanthophores are scattered widely over the flank. During metamorphosis, however, this pattern is transformed into that of the adult, which comprises several dark stripes of melanophores and iridophores that alternate with light stripes of xanthophores and iridophores. In this review, we place zebrafish relative to other model and non-model species; we review what is known about the processes of chromatophore specification, differentiation, and morphogenesis during the development of embryonic and adult pigment patterns, and we address how future studies of zebrafish will likely aid our understanding of human disease and the evolution of form.

Animals↗

PubMed2001

Characterization of duplicated zebrafish cyp19 genes.

The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.

Amino Acid Sequence↗

PubMed2005

Development of zebrafish (Danio rerio) pectoral fin musculature.

During posthatching development the fins of fishes undergo striking changes in both structure and function. In this article we examine the development of the pectoral fins from larval through adult life history stages in the zebrafish (Danio rerio), describing in detail their pectoral muscle morphology. We explore the development of muscle structure as a way to interpret the fins' role in locomotion. Genetic approaches in the zebrafish model are providing new tools for examining fin development and we take advantage of transgenic lines in which fluorescent protein is expressed in specific tissues to perform detailed three-dimensional, in vivo fin imaging. The fin musculature of larval zebrafish is organized into two thin sheets of fibers, an abductor and adductor, one on each side of an endoskeletal disk. Through the juvenile stage the number of muscle fibers increases and muscle sheets cleave into distinct muscle subdivisions as fibers orient to the developing fin skeleton. By the end of the juvenile period the pectoral girdle and fin muscles have reoriented to take on the adult organization. We find that this change in morphology is associated with a switch of fin function from activity during axial locomotion in larvae to use in swim initiation and maneuvering in adults. The examination of pectoral fins of the zebrafish highlights the yet to be explored diversity of fin structure and function in subadult developmental stages. J. Morphol. (c) 2005 Wiley-Liss, Inc.

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PubMed2004

Of lasers, mutants, and see-through brains: functional neuroanatomy in zebrafish.

Behavioral functions are carried out by localized circuits in the brain. Although this modular principle is clearly established, the boundaries of modules, and sometimes even their existence, are still debated. Zebrafish might offer distinct advantages in localizing behaviors to discrete brain regions because of the ability to visualize, record from, and lesion precisely identified populations of neurons in the brain. In addition, genetic screens in zebrafish enable the isolation of mutations that disrupt neural pathways and/or behaviors, as an alternative lesioning technique with complementary strengths to laser ablations. For example, the Mauthner cell, a large identified neuron in the hindbrain, has been postulated to be both necessary and sufficient for the execution of escapes. We discuss in this review how experiments, using laser ablations, calcium imaging, and mutants have eroded this notion. Even in a simple behavior, such as escape, many parallel pathways appear to be involved with no single one being absolutely necessary. Lesion studies and the analysis of behavioral mutants are now also beginning to elucidate the functional architecture of the zebrafish visual system. Although still in an embryonic stage, the neuroanatomy of behaviors in zebrafish has a bright future.

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PubMed1996

Mermaid, a family of short interspersed repetitive elements, is useful for zebrafish genome mapping.

A family of short interspersed repetitive elements (SINEs), designated mermaid, is present in the genomes of fish, amphibian and primates, but absent in the mouse genome. We have demonstrated that the sequences of the mermaid family are highly polymorphic in the zebrafish genome as in the human genome. We have also shown that the mermaid sequence can be used to recover zebrafish specific DNA from zebrafish-mouse cell hybrids by using mermaid-specific oligonucleotides as PCR primers. Thus, the mermaid family serves as a valuable genetic tool for the zebrafish genome mapping.

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PubMed1997

Multiplicity of neuropeptide Y receptors: cloning of a third distinct subtype in the zebrafish.

Five different receptor subtypes for neuropeptide Y (NPY) have recently been cloned in mammals. We have discovered three distinct subtypes by PCR in the zebrafish, Danio rerio, and describe here one of these called zYc. The protein sequence identity is 46-51% to mammalian subtypes Y1, Y4 and Y6 and to zebrafish Ya, i.e., the same degree of identity as these subtypes display to one another. The identity to zYb is higher, 75%, indicating that zYb and zYc share a more recent ancestor. The zYc receptor binds NPY and PYY (peptide YY) from mammals as well as zebrafish with high affinities and has a Kd of 16 pM for 125I-pPYY. The pharmacological profile is similar to, but distinct-from, mammalian Y1. zYc inhibits cAMP synthesis. This work suggests that NPY has more receptor subtypes than any other peptide that binds to G protein-coupled receptors. Work is in progress to see if the zebrafish receptors are present in mammals.

Amino Acid Sequence↗

PubMedDate not supplied

Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin.

Vitamin K-dependent gamma-carboxylation is an essential posttranslational modification required for the functional activity of coagulation proteins such as factors VII, IX, X, and prothrombin. Warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, was used in earlier work on adult zebrafish to provide evidence for the presence of vitamin K-dependent carboxylase in zebrafish. Here we demonstrate the presence of vitamin K-dependent carboxylase activity in zebrafish by directly assaying the microsomal fraction prepared from adult, unfertilized eggs, and embryos from different developmental stages. Gamma-carboxylase activity was detected both before and after fertilization of embryos and the activity levels remained relatively constant from 6 h postfertilization (hpf) through other advanced stages of development. The expression of activity in the early embryos (0-6 hpf) may be due to the presence of maternal protein since the activity was detected even in the unfertilized eggs. Gamma-carboxylase activity in the eggs as well as early embryos suggested that vitamin K-dependent carboxylase is important throughout development. The detection of vitamin K-dependent carboxylase mRNA by RT-PCR and inhibitor studies using warfarin confirmed these activity results. Further, these studies provide a basis for selecting warfarin-resistant zebrafish mutants in order to find genes regulating gamma-carboxylase activity including the yet unidentified vitamin K-epoxide reductase.

Amino Acid Sequence↗

PubMed1995

Green fluorescent fusion proteins: powerful tools for monitoring protein expression in live zebrafish embryos.

The recent development of transgenic technology in zebrafish has opened an exciting new avenue in which to explore vertebrate development. However, as in other species, the inability to easily identify live transgenic fish severely limits the potential of this promising technology. To determine whether the recently described green fluorescent protein (GFP) might provide a convenient live staining method in zebrafish, we constructed a glutathione S-transferase/GFP fusion protein (GST-GFP). GST-GFP cRNA, when injected into individual blastomeres of early zebrafish embryos, resulted in the rapid development (3 hr) of easily detectable green fluorescence which persisted for up to 4 days. GFP fluorescence was restricted to progeny of the injected cell and appeared to have no adverse effects on embryonic development despite widespread expression. Our findings demonstrate that GFP fusion proteins will provide a simple yet powerful means of monitoring production of heterologous proteins in live zebrafish.

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PubMed1995

Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish.

Zebrafish embryos have three or four identifiable primary motoneurons per hemisegment. We previously reported that, while several ventral cells initially express the zebrafish Islet-1 (Isl-1) gene, a member of the LIM/homeobox gene family, the expression of this gene becomes restricted to a single or a pair of cells slightly anterior to each segment border by 16 hr after fertilization. Double staining by in situ hybridization and immunohistochemistry strongly suggested that these cells were mainly rostral primary motoneurons. Here, we have isolated two novel zebrafish cDNA clones for more Isl-1 family genes, termed zfIsl-2 and zfIsl-3. zfIsl-2 mRNA starts to be expressed in the ventral midsegmental cells per hemisegment around 15 hr. Double labeling experiments have shown that these midsegmental cells are the caudal primary motoneuron (CaP) and its variant equivalence pair. Our results revealed the heterogeneity in the expressed genes among primary motoneurons before the fates of the primary motoneurons are irreversibly determined, and further suggest the involvement of the Isl-1 and zfIsl-2 genes in the determination of cellular identities by primary motoneurons in embryonic zebrafish. zfIsl-3 mRNA is not expressed in motoneurons but is expressed at 17 hr, mainly in the ventral myotomes. This suggests that zfIsl-3 may be involved in the regional specification of the myotome and also in target recognition by CaP. zfIsl-2 is also expressed throughout the developing eye and tectal region of the midbrain, the target for the retinal axons. In the ventral spinal cord of the spadetail mutant embryo, which has defects in the somites, the cells expressing zfIsl-2 mRNA significantly decreased in number in contrast to the increase in cells expressing Isl-1 mRNA, suggesting the influence of the somites on the expression of both genes.

Amino Acid Sequence↗

PubMed1996

Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

Animals↗

PubMed1999

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

Animals↗

PubMed2000

Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways.

The floor plate is a morphologically distinct structure of epithelial cells situated along the midline of the ventral spinal cord in vertebrates. It is a source of guidance molecules directing the growth of axons along and across the midline of the neural tube. In the zebrafish, the floor plate is about three cells wide and composed of cuboidal cells. Two cell populations can be distinguished by the expression patterns of several marker genes, including sonic hedgehog (shh) and the fork head-domain gene fkd4: a single row of medial floor plate (MFP) cells, expressing both shh and fkd4, is flanked by rows of lateral floor plate (LFP) cells that express fkd4 but not shh. Systematic mutant searches in zebrafish embryos have identified a number of genes, mutations in which visibly reduce the floor plate. In these mutants either the MFP or the LFP cells are absent, as revealed by the analysis of the shh and fkd4 expression patterns. MFP cells are absent, but LFP cells are present, in mutants of cyclops, one-eyed pinhead, and schmalspur, whose development of midline structures is affected. LFP cells are absent, but MFP cells are present, in mutants of four genes, sonic you, you, you-too, and chameleon, collectively called the you-type genes. This group of mutants also shows defects in patterning of the paraxial mesoderm, causing U- instead of V-shaped somites. One of the you-type genes, sonic you, was recently shown to encode the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. It has been shown previously that in the zebrafish shh is required for the induction of LFP cells, but not for the development of MFP cells. This conclusion is supported by the finding that injection of shh RNA causes an increase in the number of LFP, but not MFP cells. Embryos mutant for iguana, detour, and umleitung share the lack of LFP cells with you-type mutants while somite patterning is not severely affected. In mutants that fail to develop a notochord, MFP cells may be present, but are always surrounded by LFP cells. These data indicate that shh, expressed in the notochord and/or the MFP cells, induces the formation of LFP cells. In embryos doubly mutant for cyclops (cyc) and sonic you (syu) both LFP and MFP cells are deleted. The number of primary motor neurons is strongly reduced in cyc;syu double mutants, while almost normal in single mutants, suggesting that the two different pathways have overlapping functions in the induction of primary motor neurons.

Animals↗

PubMed2002

Zebrafish SPI-1 (PU.1) marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning.

The mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning.

Animals↗

PubMed2001

Identification and characterization of a novel fibronectin in zebrafish.

Sequence analysis of zebrafish fibronectin (FN) cDNAs indicates that at least two forms of the protein exist in fish. One form (FN1) is very similar to FNs identified in other vertebrates possessing 12 type I, 2 type II, and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1 contains the RGD cell adhesion site in type III(10) and a second cell-binding site (LDV) in the V region. In addition to this conserved form of FN, a novel truncated form of zebrafish FN (FN2) was identified. The predicted structure of FN2 is identical to FN1 at the N-terminal region possessing 9 type I, 2 type II, and the first 3 type III repeats. Following III(3), FN2 contains a unique 20-amino-acid C-terminal tail that is different from the C-terminus of FN1, lacking the two cysteines that are usually involved in the formation of interchain disulfide bonds. Genomic sequence analysis has revealed that FN2 is generated by an alternative RNA splicing pattern that has not been described for FN in other organisms. Reverse transcription-polymerase chain reaction analysis and RNase protection assays reveal that FN2 mRNA is present in the zebrafish embryo throughout development as well as in cultures of an established liver cell line. Experiments conducted with recombinant FN2 synthesized in insect cells demonstrate that the protein promotes the attachment and spreading of fish embryo cells in culture.

Alternative Splicing↗

PubMed2000

Centromere-linked microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 of zebrafish (Danio rerio).

A large number of interesting mutations affecting development and organogenesis have been identified through genetic screens in zebrafish. Mapping of these mutations to a chromosomal region can be rapidly accomplished using half-tetrad analysis. However, knowledge of centromere-linked markers on every chromosome is essential to this mapping method. Centromeres on all 25 linkage groups have been mapped on the RAPD zebrafish genetic map. However, species specificity and the lack of codominance make RAPD markers less practical for mapping than microsatellite-based markers. On the microsatellite-based genetic map, centromere-linked markers have been identified for 19 linkage groups. No direct evidence has been published linking microsatellite markers to the centromeres of linkage groups 3, 4, 6, 7, 13, and 20. Therefore, we compared the microsatellite-based genetic map with the RAPD map to identify markers most likely linked to the centromeres of these 6 linkage groups. These candidate markers were tested for potential centromere linkage using four panels of half-tetrad embryos derived by early-pressure treatment of eggs from four different female zebrafish. We have identified microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 to within 1.7 cM of their centromeres. These markers will greatly facilitate the rapid mapping of mutations in zebrafish by half-tetrad analysis.

Animals↗

PubMed2002

A genetic, deletion, physical, and human homology map of the long fin region on zebrafish linkage group 2.

Mutation of the gene long fin causes overgrowth of zebrafish fins. Thus, molecular identification of the gene long fin may reveal the mechanisms involved in normal growth control. We have therefore developed genetic and physical maps of the corresponding region on linkage group 2 (LG2). A single deletion allele (lof(jg)(61)) of the long fin gene was also generated. Examination of this deletion for the presence or absence of ESTs independently mapped to LG2 revealed a contiguous deletion of SSLP, STS, and gene-based markers spanning a physical distance of approximately 500 kb, including at least 10 zebrafish genes. Human orthologs of the zebrafish genes in the long fin region were identified and revealed two synteny segments from human chromosome 1 (Hsa1) and Hsa19. Homology searching for additional genes linked to the human orthologs revealed one additional gene in the long fin deletion region. Thus, our development of the genetic, physical, deletion, and human homology maps of the long fin region provides one of the first high-resolution comparisons of a zebrafish region with a homologous human region, and facilitates the molecular identification of the long fin gene.

Animals↗

PubMed2002

Patterning the zebrafish central nervous system.

We have described the formation of the zebrafish central nervous system. The spinal cord has the simplest organization and was considered first, followed by the forebrain, midbrain and hindbrain. We have discussed many studies that have revealed the molecular mechanisms, including extrinsic signals and intrinsic responses to them, underlying the establishment of nervous system regions and the wide diversity of neuronal cell types of which they are comprised. Wherever possible, we have tried to compare what has been learned from zebrafish with what is known in other vertebrate species. The simplicity of the developing nervous system makes zebrafish embryos particularly amenable to studies of nervous system development. Thus, many aspects of nervous system patterning that were unknown from other vertebrates have been revealed by studies in zebrafish. However, the relationship between embryonic and adult nervous system morphology is still not entirely clear and remains an important avenue for further studies.

Animals↗

PubMed2002

The zebrafish eye: developmental and genetic analysis.

In this review, we have attempted to cover all the major points of zebrafish eye development, and have found that, for the most part, it has much in common with other eyes, in both vertebrates and the fly. In addition to the confirmation and extension of earlier studies, however, the work on zebrafish has provided some new insights that should be assessed for their applicability to the development of other vertebrates. Among these are the modulated cellular proliferation in the optic vesicle, the complex spatiotemporal pattern of central retinal neurogenesis, the emergence of spatial order among the photoreceptors, the genetic controls of cell fates, and the genetic mechanisms underlying retinal stratification. Substantial though it is, this contribution will grow rapidly in the next few years as the advances of zebrafish genetics are accelerated by progress of genomics, especially the zebrafish genome project.

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