Banded chromosomes and the zebrafish karyotype.
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Animals↗
Biomedical subjects
J H Postlethwait
Publications and source records attributed to J H Postlethwait.
Isolation of zebrafish gdf7 and comparative genetic mapping of genes belonging to the growth/differentiation factor 5, 6, 7 subgroup of the TGF-beta superfamily.
The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.
Amino Acid Sequence↗
Cloning of two loci for synapse protein Snap25 in zebrafish: comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage.
Synaptosome-associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage.
Amino Acid Sequence↗
Zebrafish hox clusters and vertebrate genome evolution.
HOX genes specify cell fate in the anterior-posterior axis of animal embryos. Invertebrate chordates have one HOX cluster, but mammals have four, suggesting that cluster duplication facilitated the evolution of vertebrate body plans. This report shows that zebrafish have seven hox clusters. Phylogenetic analysis and genetic mapping suggest a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-finned and lobe-finned fishes but before the teleost radiation. Thus, teleosts, the most species-rich group of vertebrates, appear to have more copies of these developmental regulatory genes than do mammals, despite less complexity in the anterior-posterior axis.
Animals↗
Gene mapping in zebrafish using single-strand conformation polymorphism analysis.
To exploit fully the power of the zebrafish system as a model for vertebrate development, it will be necessary to develop efficient tools for genomic analysis. In this report we have tested whether single-strand conformation polymorphism analysis (SSCP) can be utilized for gene mapping in zebrafish. Over 100 primer pairs derived from noncoding regions of known genes and partially characterized cDNAs were analyzed, and a polymorphism frequency of approximately 50% was detected in zebrafish strains used for genetic mapping studies. A subset of these polymorphic cDNAs was localized on the zebrafish map. SSCP thus represents an efficient strategy for mapping transcribed sequences with a high resolution in the zebrafish genome, which will facilitate the integration of existing zebrafish framework maps, the generation of a zebrafish EST map, and the application of alternative gene localization strategies such as comparative mapping.
3' Untranslated Regions↗
Mutant rescue by BAC clone injection in zebrafish.
Genes essential for vertebrate body plan specification, organ development, and organ function are likely to be shared between mammals and zebrafish, but only in zebrafish have large-scale, genome-wide mutagenesis screens been conducted to isolate embryonic lethal mutations. Discovering the roles played by these disrupted genes requires their molecular characterization, which would be facilitated by assaying large cloned genomic DNAs for their potential to rescue mutant phenotypes. Here we demonstrate that bacterial artificial chromosomes can rescue the phenotype of floating head (flh) mutants. Homozygous flh embryos lack a differentiated notochord and have a reduced, discontinuous floor plate. Mutant embryos injected with genomic clones containing the flh+ gene often had stretches of several to many notochord cells overlaid by a row of floor-plate cells. In contrast, control mutant embryos injected with artificial chromosomes lacking the flh+ gene failed to form notochord. We conclude that the injection of large-insert genomic clones will speed the isolation of zebrafish genes disrupted by mutation and hence the identification of gene functions necessary for development of vertebrate embryos.
Animals↗
The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis.
In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.
Adaptor Proteins, Signal Transducing↗
SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish.
SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation.
Amino Acid Sequence↗
Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos.
To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.
Amino Acid Sequence↗
Vertebrate genome evolution and the zebrafish gene map.
In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.
Animals↗
Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome.
Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.
Alleles↗
Genetic interactions in zebrafish midline development.
Mutational analyses have shown that the genes no tail (ntl, Brachyury homolog), floating head (flh, a Not homeobox gene), and cyclops (cyc) play direct and essential roles in the development of midline structures in the zebrafish. In both ntl and flh mutants a notochord does not develop, and in cyc mutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent in cyc;flh double mutants than in either cyc or flh single mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects in flh;ntl double mutants are not additive, but are most often similar to those in ntl single mutants. This finding reveals that loss of ntl function can suppress phenotypic defects due to mutation at flh, and we interpret it to mean that the wild-type allele of ntl (ntl+) functions upstream to flh in a regulatory hierarchy. Loss of function of ntl also strongly suppresses the floor plate deficiency in cyc mutants, for we found trunk floor plate to be present in cyc;ntl double mutants. From these findings we propose that ntl+ plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development.
Animals↗
Mapping of mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio.
The mammalian major histocompatibility complex (Mhc) consists of three closely linked regions, I, II, and III, occupying a single chromosomal segment. The class I loci in region I and the class II loci in region II are related in their structure, function, and evolution. Region III, which is intercalated between regions I and II, contains loci unrelated to the class I and II loci, and to one another. There are indications that a similar Mhc organization exists in birds and amphibians. Here, we demonstrate that in the zebrafish (Danio rerio), a representative of the teleost fishes, the class II loci are divided between two linkage groups which are distinct from the linkage group containing the class I loci. The beta2-microglobulin-encoding gene is loosely linked to one of the class II loci. The gene coding for complement factor B, which is one of the region III genes in mammals, is linked neither to the class I nor to the class II loci in the zebrafish. These results, combined with preliminary data suggesting that the class I and class II regions in another order of teleost fish are also in different linkage groups, indicate that close linkage of the two regions is not necessary either for regulation of expression or for co-evolution of the class I and class II loci. They also raise the question of whether linkage of the class I and class II loci in tetrapods is a primitive or derived character.
Animals↗
Zebrafish genomics: from mutants to genes.
Exquisite embryonic lethal mutations have been isolated in hundreds of genes necessary for zebrafish development. Analysis of this resource promises to enhance our understanding of the molecular genetic mechanisms of vertebrate development. This review discusses the state of the zebrafish genome project and the genetic trickery that can expedite molecular isolation of genes disrupted by these mutations.
Animals↗
MeV-ion microprobe analyses of whole Drosophila suggest that zinc and copper accumulation is regulated storage not deposit excretion.
We examined Drosophila spp. using a penetrative ion microprobe technique that allows us to quantify element contents in whole organs and organisms. Comparatively non-penetrative techniques, such as electron microscopy, could not have been used to make many of these measurements because material is lost during sectioning. We found that zinc was accumulated predominantly within a single organ: in the main segments of both the anterior and posterior Malpighian tubules. In contrast to zinc, iron and copper were more generally distributed throughout the body. Zinc concentrations as high as 2.8 % of dry mass were measured in cell-sized volumes of the Malpighian tubules. The large quantities of zinc (approximately 2x10(-8) g in 8-day-old male adults) were sequestered by an unidentified mechanism. We found less than 1 % of the estimated amount of consumed zinc and copper in the abdomen of flies fed food containing several hundred parts per million dry mass of these metals. Our results are inconsistent with the detoxification hypothesis that predicts that a large proportion of the heavy metals passing through the gut are absorbed and stored permanently. We found for both zinc and copper that the quantity in the abdomen was not proportional to the concentration of these metals in the consumed food but was, instead, relatively invariant. For these reasons, we suggest that regulated biological availability, not detoxification, may be the primary benefit of zinc and copper storage.
Animals↗
Centromere-linkage analysis and consolidation of the zebrafish genetic map.
The ease of isolating mutations in zebrafish will contribute to an understanding of a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the number of mapped PCR-based markers to 652, and consolidated the number of linkage groups to the number of chromosomes. This work makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads.
Animals↗
A homeobox gene essential for zebrafish notochord development.
The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.
Amino Acid Sequence↗
Expression of a type II collagen gene in the zebrafish embryonic axis.
To understand the hierarchy of developmental controls underlying axis specification in vertebrate embryos, it is helpful to identify relationships between regulatory molecules and the genes that give axial cells their differentiated phenotypes. This work reports the cloning and expression pattern of one of these differentiation genes, a type II collagen (col2a1) gene from the zebrafish Danio rerio. Along the embryonic axis, col2a1 is expressed dynamically in three rows that are each a single cell wide: the notochord and the rows of cells immediately dorsal and ventral to it--the floor plate of the central nervous system, and the hypochord. In addition, col2a1 is expressed in the pharyngeal arches, the epithelium of the otic capsule, and in the mesenchyme of the neurocranium. Experiments probed the expression pattern of col2a1 relative to that of known or potential regulators of axis development, including axial, sonic hedgehog, twist, and cyclops. The results showed that the spatial and temporal pattern of col2a1 expression in axial mesoderm follows the expression of twist closer than other genes tested. In cyclops embryos, which lack an intact floor plate, col2a1 expression was usually low, but not missing in cells in the ventral spinal cord. Because col2a1 expression reveals abnormalities in the notochord of cyclopsb16 embryos, and less col2a1-expressing mesenchyme accumulates rostral to the notochord in cyclops embryos, the effects of the cyclopsb16 mutation are not confined to the central nervous system.
Animals↗