miRNA Nomenclature: A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants.

High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of miRNAs to encompass this diversity. We then review nomenclature guidelines for miRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations (i.e., isomiRs) in the analysis of small RNA sequencing experiments.

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.

The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension.

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

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.

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.

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.

Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages.

Bradykinin acts through two receptor subtypes in mammals and generates a variety of responses including pain, inflammation and hypotension. The evolutionary history of the bradykinin system has been unclear due to shortage of information outside mammals. We describe here two receptor subtypes and the bradykinin precursor in three species of bony fish (the zebrafish Danio rerio, the Japanese pufferfish Takifugu rubripes, and the green spotted pufferfish Tetraodon nigroviridis) and chicken and analyze the relationships to mammals by a combination of phylogeny, conserved synteny and exon-intron organization. All of these species have two receptor genes located close to each other in a tandem formation, with the B2 gene 5' to the B1 gene, in chromosomal regions displaying conserved synteny between the species (albeit conservation of synteny in zebrafish is still unclear due to poor genome assembly). The evolutionary rate differs between the two genes as well as between lineages leading to differing pharmacological properties for both B1 and B2 across vertebrate classes. Also the bradykinin precursor gene was identified in all of these species in a chromosome region with conserved synteny. The tissue distribution of mRNA in T. rubripes is similar for B1 and B2, suggesting more similar regulation for the two genes than in mammals. In conclusion, the receptor tandem duplication predates the divergence of ray-finned fish and tetrapods and no additional duplicates of the receptors or bradykinin seem to have survived the ray-finned fish tetraploidization.

Assignment of zebrafish genetic linkage groups to chromosomes.

In this report the zebrafish genetic linkage groups are assigned to specific chromosomes using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group (LG). Chromosomes were identified using a combination of relative size and arm ratios. The largest genetic maps generally corresponded to the largest chromosomes, but genetic recombination tended to be elevated in the smaller chromosomes and near telomeres. Large insert clones containing genes near telomeres often hybridized to telomeres of multiple chromosome pairs, suggesting the presence of shared subtelomeric repetitive DNAs near telomeres. Evidence from comparative gene mapping in medaka, zebrafish, pufferfish, and humans suggests that the linkage groups of these species have the content of duplicate proto-chromosomes. However, these duplicate linkage groups are not associated with chromosomes of similar size or morphology. This suggests that considerable chromosome restructuring occurred subsequent to the genome duplication in teleosts.

Development in genetic mosaics of aristapedia, a homoeotic mutant of Drosophila melanogaster.

Development of the homoeotic mutation, aristapedia (ss(a)), was investigated by means of genetic mosaics. The wild-type alleles of aristapedia and the bristle markers yellow, singed, and forked were removed from cells at different times in development by X-ray induced somatic crossing-over. The phenotype of the resulting clones was examined in order to ascertain whether it was leg or antenna. The y sn f; ss(a) clones showed a leg phenotype if induced before the mid-third instar, but showed an antennal phenotype if induced after this time. Late non-expression of ss(a) may be due either to an influence of surrounding ss(+) tissues on the small ss(a) clones, or to a persistence of the effect of ss(+) for one or two cell generations after it is removed from a cell line.

Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.

Analysis of meiotic tetrads is routinely used to determine genetic linkage in various fungi. Here we apply tetrad analysis to the study of genetic linkage in a vertebrate. The half-tetrad genotypes of gynogenetic diploid zebrafish produced by early-pressure (EP) treatment were used to investigate the linkage relationships of two recessive pigment pattern mutations, leopard (leo) and rose (ros). The results showed that ros is tightly linked to its centromere and leo maps 31 cM from its centromere. Analysis of half-tetrads segregating for ros and leo in repulsion revealed no homozygous ros individuals among 32 homozygous leo half-tetrads--i.e., a parental ditype (PD) to nonparental ditype (NPD) ratio of 32:0. This result shows that ros is linked to leo, a mutation previously mapped to Linkage Group I. Investigation of PCR-based DNA polymorphisms on Linkage Group I confirmed the location of ros near the centromere of this linkage group. We propose an efficient, generally useful method to assign new mutations to a linkage group in zebrafish by determining which of 25 polymerase chain reaction (PCR)-based centromere markers shows a significant excess of PD to NPD in half-tetrad fish.

Preservation of duplicate genes by complementary, degenerative mutations.

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

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.

Functional characterization and genetic mapping of alk8.

The novel type I TGFbeta family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.

The BMP-related protein radar: a maintenance factor for dorsal neuroectoderm cells?

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.

A recessive mutation leading to vertebral ankylosis in zebrafish is associated with amino acid alterations in the homologue of the human membrane-associated guanylate kinase DLG3.

We describe the characterization of the zebrafish homologue of the human gene DLG3. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fishes. Insertion and mutation have both been mapped to chromosome 12, in a segment which is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain.

Generation of a P1 artificial chromosome library of the Southern pufferfish.

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.

Two variants of zebrafish p100 are expressed during embryogenesis and regulated by Nodal signaling.

Human p100 protein was first identified as a transcriptional coactivator of Epstein-Barr virus nuclear antigen 2, and has been shown to be a coactivator of other cellular transactivators. Its roles in development of vertebrate embryos, however, have not been reported. We have identified a zebrafish ortholog of the human p100 coactivator. The zebrafish p100 transcript is processed to two alternative variants, long and short forms, referred to as p100L and p100S, respectively. Both GFP-p100L and GFP-p100S fusion proteins are located in the cytoplasm of transfected culture cells and microinjected embryonic cells. Analysis of transcripts with Northern blots revealed the presence of p100L and lower amounts of p100S mRNAs from the one-cell stage throughout the life cycle. Whole-mount in situ hybridization shows that p100L and p100S share the same spatiotemporal expression pattern. Their zygotic expression is initially restricted to axial mesoderm precursors during gastrulation, and then spreads over other tissues during segmentation, and finally is constrained to some internal organs at day 5. We also find that Nodal signaling is essential for the zygotic expression of p100. These studies pave the way to understanding in depth the role of p100 during vertebrate embryogenesis.

The humoral antibacterial response of Drosophila adults.

Hemolymph from a normal adult Drosophila melanogaster lacks factors that block the growth of Escherichia coli, but hemolymph from a fly previously inoculated with Enterobacter cloacae inhibits bacterial growth. Antibacterial activity appears within two hours after inoculation, and is still detectable sixty days later. Activity is potent, and can be detected in as little as a quarter of the hemolymph from a single inoculated male fly. After inoculation, at least eight new polypeptides not of bacterial origin appear in hemolymph with a time course similar to the appearance of antibacterial activity; these are called Antibacterial Response Polypeptides, or ARs. The most prominent polypeptides are AR24, AR22, and AR19 with molecular weights of about 24, 22, and 19 kilodaltons (kd). Other bands with as much as 75 kd and as little as 5 kd were also found. Electrophoresis of active hemolymph under non-denaturing conditions, and isoelectric focusing separate several protein species that block bacterial growth (Antibacterial Proteins, or ABs); one AB is neutral (AB7.1) and three are basic (AB8.7, AB9.0 and AB9.2). Two dimensional gels show that AR24, AR22 and AR19 have pIs identical to the basic antibacterial proteins. Radiolabelling experiments proved that the ARs were synthesized de novo after bacterial inoculation. ARs in six species of Drosophila showed fundamentally similar electrophoretic patterns.

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.