A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

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.

A genetic linkage map for the zebrafish.

To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

The zebrafish organizer.

Signals from the organizer play a crucial role in patterning the vertebrate embryo. Recent molecular analysis of zebrafish mutations has established an essential role for BMP2 and chordin in organizer function and has identified one-eyed pinhead as a novel EGF-like gene involved in prechordal plate and endoderm formation. In addition, embryological studies have suggested that the zebrafish organizer is induced by extraembryonic cues and have defined two novel organizing centers that pattern the nervous system along the anterior-posterior axes.

Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish.

The vertebrate body plan arises during gastrulation, when morphogenetic movements form the ectoderm, mesoderm, and endoderm. In zebrafish, mesoderm and endoderm derive from the marginal region of the late blastula, and cells located nearer the animal pole form the ectoderm [1]. Analysis in mouse, Xenopus, and zebrafish has demonstrated that Nodal-related proteins, a subclass of the TGF-beta superfamily, are essential for mesendoderm development [2], but previous mutational studies have not established whether Nodal-related signals control fate specification, morphogenetic movements, or survival of mesendodermal precursors. Here, we report that Nodal-related signals are required to allocate marginal cells to mesendodermal fates in the zebrafish embryo. In double mutants for the zebrafish nodal-related genes squint (sqt) and cyclops (cyc) [3] [4] [5], dorsal marginal cells adopt neural fates, whereas in wild-type embryos, cells at this position form endoderm and axial mesoderm. Involution movements characteristic of developing mesendoderm are also blocked in the absence of Nodal signaling. Because it has been proposed [6] that inhibition of Nodal-related signals promotes the development of anterior neural fates, we also examined anteroposterior organization of the neural tube in sqt;cyc mutants. Anterior trunk spinal cord is absent in sqt;cyc mutants, despite the presence of more anterior and posterior neural fates. These results demonstrate that nodal-related genes are required for the allocation of dorsal marginal cells to mesendodermal fates and for anteroposterior patterning of the neural tube.

Fast1 is required for the development of dorsal axial structures in zebrafish.

Nodal-related signals comprise a subclass of the transforming growth factor-beta (TGF-beta) superfamily and regulate key events in vertebrate embryogenesis, including mesoderm formation, establishment of left-right asymmetry and neural patterning [1-8]. Nodal ligands are thought to act with EGF-CFC protein co-factors to activate activin type I and II or related receptors, which phosphorylate Smad2 and trigger nuclear translocation of a Smad2/4 complex [8-12]. The winged-helix transcription factor forkhead activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2 [12-20]. Xenopus Fast-1 is thought to function as a transcriptional effector of Nodal signals during mesoderm formation [17], but no mutations in the Fast-1 gene have been identified. We report the identification of the zebrafish fast1 gene and show that it is disrupted in schmalspur (sur) mutants, which have defects in the development of dorsal midline cell types and establishment of left-right asymmetry [21-25]. We find that prechordal plate and notochord are strongly reduced in maternal-zygotic sur mutants, whereas other mesendodermal structures are present - a less severe phenotype than that caused by complete loss of Nodal signaling. These results show that fast1 is required for development of dorsal axial structures and left-right asymmetry, and suggest that Nodal signals act through Fast1-dependent and independent pathways.

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.

Analysis of chromosomal rearrangements induced by postmeiotic mutagenesis with ethylnitrosourea in zebrafish.

Mutations identified in zebrafish genetic screens allow the dissection of a wide array of problems in vertebrate biology. Most screens have examined mutations induced by treatment of spermatogonial (premeiotic) cells with the chemical mutagen N-ethyl-N-nitrosourea (ENU). Treatment of postmeiotic gametes with ENU induces specific-locus mutations at a higher rate than premeiotic regimens, suggesting that postmeiotic mutagenesis protocols could be useful in some screening strategies. Whereas there is extensive evidence that ENU induces point mutations in premeiotic cells, the range of mutations induced in postmeiotic zebrafish germ cells has been less thoroughly characterized. Here we report the identification and analysis of five mutations induced by postmeiotic ENU treatment. One mutation, snh(st1), is a translocation involving linkage group (LG) 11 and LG 14. The other four mutations, oep(st2), kny(st3), Df(LG 13)(st4), and cyc(st5), are deletions, ranging in size from less than 3 cM to greater than 20 cM. These results show that germ cell stage is an important determinant of the type of mutations induced. The induction of chromosomal rearrangements may account for the elevated frequency of specific-locus mutations observed after treatment of postmeiotic gametes with ENU.

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.

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.

Nodal signaling and the zebrafish organizer.

Systematic genetic screens in zebrafish have led to the discovery of mutations that affect organizer function and development. The molecular isolation and phenotypic analysis of the affected genes have revealed that TGF-beta signals of the Nodal family play a key role in organizer formation. The activity of the Nodal signals Cyclops and Squint is regulated extracellularly by the EGF-CFC cofactor One-eyed Pinhead and by antagonists belonging to the Lefty family of TGF-beta molecules. In the absence of Nodal signaling, the fate of cells in the organizer is transformed from dorsal mesoderm to neural ectoderm. Differential Nodal signaling also patterns the organizer along the anterior-posterior axis, with high levels required for anterior cell fates and lower levels for posterior fates. In addition, Nodal signaling cooperates with the homeodomain transcription factor Bozozok in organizer formation and neural patterning. The combination of genetic, molecular and embryological approaches in zebrafish has thus provided a framework to understand the mechanisms underlying organizer development.

Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms.

In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.

Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning.

Zebrafish you-too (yot) mutations interfere with Hedgehog (Hh) signaling during embryogenesis. Using a comparative synteny approach, we isolated yot as a zinc finger transcription factor homologous to the Hh target gli2. Two alleles of yot contain nonsense mutations resulting in carboxy-terminally truncated proteins. In addition to causing defects in midline development, muscle differentiation, and retinal axon guidance, yot mutations disrupt anterior pituitary and ventral forebrain differentiation. yot mutations also cause ectopic lens formation in the ventral diencephalon. These findings reveal that truncated zebrafish Gli2 proteins interfere with Hh signaling necessary for differentiation and axon guidance in the ventral forebrain.

Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation.

The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. We report the molecular isolation of oep using a positional cloning approach. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation.

Nodal signaling patterns the organizer.

Spemann's organizer plays an essential role in patterning the vertebrate embryo. During gastrulation, organizer cells involute and form the prechordal plate anteriorly and the notochord more posteriorly. The fate mapping and gene expression analyses in zebrafish presented in this study reveal that this anteroposterior polarity is already initiated in the organizer before gastrulation. Prechordal plate progenitors reside close to the blastoderm margin and express the homeobox gene goosecoid, whereas notochord precursors are located further from the margin and express the homeobox gene floating head. The nodal-related genes cyclops and squint are expressed at the blastoderm margin and are required for prechordal plate and notochord formation. We show that differential activation of the Nodal signaling pathway is essential in establishing anteroposterior pattern in the organizer. First, overexpression of cyclops and squint at different doses leads to the induction of floating head at low doses and the induction of both goosecoid and floating head at higher doses. Second, decreasing Nodal signaling using different concentrations of the antagonist Antivin inhibits goosecoid expression at low doses and blocks expression of both goosecoid and floating head at higher doses. Third, attenuation of Nodal signaling in zygotic mutants for the EGF-CFC gene one-eyed pinhead, an essential cofactor for Nodal signaling, leads to the loss of goosecoid expression and expansion of floating head expression in the organizer. Concomitantly, cells normally fated to become prechordal plate are transformed into notochord progenitors. Finally, activation of Nodal signaling at different times suggests that prechordal plate specification requires sustained Nodal signaling, whereas transient signaling is sufficient for notochord development. Together, these results indicate that differential Nodal signaling patterns the organizer before gastrulation, with the highest level of activity required for anterior fates and lower activity essential for posterior fates.

Programmed cell death in the Drosophila CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform.

At adult emergence, the ventral CNS of Drosophila shows a group of approximately 300 neurons, which are unique in that they express 10-fold higher levels of the A isoform of the ecdysone receptor (EcR-A) than do other central neurons. This expression pattern is established early in metamorphosis and persists throughout the remainder of the pupal stage. Although these cells represent a heterogeneous group of neurons, they all share the same fate of undergoing rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Treatment of flies with 20-hydroxyecdysone blocks the death of the cells, but only if given at least 3 hours before the normal time of degeneration. The correlation of a unique pattern of receptor isoform expression with a particular steroid-regulated fate suggests that variations in the pattern of receptor isoform expression may serve as important switches during development.

bozozok and squint act in parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish.

In vertebrate embryos, maternal (beta)-catenin protein activates the expression of zygotic genes that establish the dorsal axial structures. Among the zygotically acting genes with key roles in the specification of dorsal axial structures are the homeobox gene bozozok (boz) and the nodal-related (TGF-(beta) family) gene squint (sqt). Both genes are expressed in the dorsal yolk syncytial layer, a source of dorsal mesoderm inducing signals, and mutational analysis has indicated that boz and sqt are required for dorsal mesoderm development. Here we examine the regulatory interactions among boz, sqt and a second nodal-related gene, cyclops (cyc). Three lines of evidence indicate that boz and sqt act in parallel to specify dorsal mesoderm and anterior neuroectoderm. First, boz requires sqt function to induce high levels of ectopic dorsal mesoderm, consistent with sqt acting either downstream or in parallel to boz. Second, sqt mRNA is expressed in blastula stage boz mutants, indicating that boz is not essential for activation of sqt transcription, and conversely, boz mRNA is expressed in blastula stage sqt mutants. Third, boz;sqt double mutants have a much more severe phenotype than boz and sqt single mutants. Double mutants consistently lack the anterior neural tube and axial mesoderm, and ventral fates are markedly expanded. Expression of chordin and noggin1 is greatly reduced in boz;sqt mutants, indicating that the boz and sqt pathways have overlapping roles in activating secreted BMP antagonists. In striking contrast to boz;sqt double mutants, anterior neural fates are specified in boz;sqt;cyc triple mutants. This indicates that cyc represses anterior neural development, and that boz and sqt counteract this repressive function. Our results support a model in which boz and sqt act in parallel to induce dorsalizing BMP-antagonists and to counteract the repressive function of cyc in neural patterning.

Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development.

In insects, the ecdysteroids act to transform the CNS from its larval to its adult form. A key gene in this response is the ecdysone receptor (EcR), which has been shown in Drosophila to code for 3 protein isoforms. Two of these isoforms, EcR-A and EcR-B1, are prominently expressed in the CNS and we have used isoform-specific antibodies to examine their fluctuations through postembryonic life. EcR expression at the onset of metamorphosis is extremely diverse but specific patterns of EcR expression correlate with distinct patterns of steroid response. Most larval neurons show high levels of EcR-B1 at the start of metamorphosis, a time when they lose larval features in response to ecdysteroids. Earlier, during the larval molts, the same cells have no detectable receptors and show no response to circulating ecdysteroids; later, during the pupal-adult transformation, they switch to EcR-A expression and respond by maturing to their adult form. During the latter period, a subset of the larval neurons hyperexpress EcR-A and these cells are fated to die after the emergence of the adult. The stem cells for the imaginal neurons show prominent EcR-B1 expression during the last larval stage correlated with their main proliferative period. Most imaginal neurons, by contrast, express only EcR-A when they subsequently initiate maturation at the start of metamorphosis. The imaginal neurons of the mushroom bodies are unusual amongst imaginal neurons in expressing the B1 isoform at the start of metamorphosis but they also show regressive changes at this time as they lose their larval axons. Imaginal neurons of the optic lobe show a delayed expression of EcR-B1 through the period when cell-cell interactions are important for establishing connections within this region of the CNS. Overall, the appearance of the two receptor isoforms in cells correlates with different types of steroid responses: EcR-A predominates when cells are undergoing maturational responses whereas EcR-B1 predominates during proliferative activity or regressive responses. The heterogeneity of EcR expression at the start of metamorphosis presumably reflects the diverse origins and requirements of the neurons that nevertheless are all exposed to a common hormonal signal.

The EGF-CFC protein one-eyed pinhead is essential for nodal signaling.

The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.

Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms.

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.