Expression of olfactory receptor genes in non-olfactory tissues in the developing and adult zebrafish.

Since the discovery of olfactory receptor (OR) genes, their expression in non-olfactory tissues have been reported in rodents and humans. For example, mouse OR23 (mOR23) is expressed in sperm and muscle cells and has been proposed to play a role in chemotaxis and muscle migration, respectively. In addition, mouse mesencephalic dopaminergic neurons express various ORs, which respond to corresponding ligands. As the OR genes comprise the largest multigene family of G protein-coupled receptors in vertebrates (over 400 genes in human and 1000 in rodents), it has been difficult to categorize the extent of their diverse expression in non-olfactory tissues making it challenging to ascertain their function. The zebrafish genome contains significantly fewer OR genes at around 140 genes, and their expression pattern can be easily analyzed by carrying out whole mount in situ hybridization (ISH) assay in larvae. In this study, we found that 31 out of 36 OR genes, including or104-2, or108-1, or111-1, or125-4, or128-1, or128-5, 133-4, or133-7, or137-3 are expressed in various tissues, including the trunk, pharynx, pancreas and brain in the larvae. In addition, some OR genes are expressed in distinct brain regions such as the hypothalamus and the habenula in a dynamic temporal pattern between larvae, juvenile and adult zebrafish. We further confirmed that OR genes are expressed in non-olfactory tissues by RT-PCR in larvae and adults. These results indicate tight regulation of OR gene expression in the brain in a spatial and temporal manner and that the expression of OR genes in non-olfactory tissues are conserved in vertebrates. This study provides a framework to start investigating the function of ORs in the zebrafish brain.

Is the species flock concept operational? The Antarctic shelf case.

There has been a significant body of literature on species flock definition but not so much about practical means to appraise them. We here apply the five criteria of Eastman and McCune for detecting species flocks in four taxonomic components of the benthic fauna of the Antarctic shelf: teleost fishes, crinoids (feather stars), echinoids (sea urchins) and crustacean arthropods. Practical limitations led us to prioritize the three historical criteria (endemicity, monophyly, species richness) over the two ecological ones (ecological diversity and habitat dominance). We propose a new protocol which includes an iterative fine-tuning of the monophyly and endemicity criteria in order to discover unsuspected flocks. As a result nine « full ¼ species flocks (fulfilling the five criteria) are briefly described. Eight other flocks fit the three historical criteria but need to be further investigated from the ecological point of view (here called "core flocks"). The approach also shows that some candidate taxonomic components are no species flocks at all. The present study contradicts the paradigm that marine species flocks are rare. The hypothesis according to which the Antarctic shelf acts as a species flocks generator is supported, and the approach indicates paths for further ecological studies and may serve as a starting point to investigate the processes leading to flock-like patterning of biodiversity.

In situ gene mapping of two genes supports independent evolution of sex chromosomes in cold-adapted Antarctic fish.

Two genes, that is, 5S ribosomal sequences and antifreeze glycoprotein (AFGP) genes, were mapped onto chromosomes of eight Antarctic notothenioid fish possessing a X1X1X2X2/X1X2Y sex chromosome system, namely, Chionodraco hamatus and Pagetopsis macropterus (family Channichthyidae), Trematomus hansoni, T. newnesi, T. nicolai, T. lepidorhinus, and Pagothenia borchgrevinki (family Nototheniidae), and Artedidraco skottsbergi (family Artedidraconidae). Through fluorescence in situ hybridization (FISH), we uncovered distinct differences in the gene content of the Y chromosomes in the eight species, with C. hamatus and P. macropterus standing out among others in bearing 5S rDNA and AFGP sequences on their Y chromosomes, respectively. Both genes were absent from the Y chromosomes of any analyzed species. The distinct patterns of Y and non-Y chromosome association of the 5S rDNA and AFGP genes in species representing different Antarctic fish families support an independent origin of the sex heterochromosomes in notothenioids with interesting implications for the evolutionary/adaptational history of these fishes living in a cold-stable environment.

A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs.

BACKGROUND: The Nile tilapia (Oreochromis niloticus) is the second most farmed fish species worldwide. It is also an important model for studies of fish physiology, particularly because of its broad tolerance to an array of environments. It is a good model to study evolutionary mechanisms in vertebrates, because of its close relationship to haplochromine cichlids, which have undergone rapid speciation in East Africa. The existing genomic resources for Nile tilapia include a genetic map, BAC end sequences and ESTs, but comparative genome analysis and maps of quantitative trait loci (QTL) are still limited. RESULTS: We have constructed a high-resolution radiation hybrid (RH) panel for the Nile tilapia and genotyped 1358 markers consisting of 850 genes, 82 markers corresponding to BAC end sequences, 154 microsatellites and 272 single nucleotide polymorphisms (SNPs). From these, 1296 markers could be associated in 81 RH groups, while 62 were not linked. The total size of the RH map is 34,084 cR(3500) and 937,310 kb. It covers 88% of the entire genome with an estimated inter-marker distance of 742 Kb. Mapping of microsatellites enabled integration to the genetic map. We have merged LG8 and LG24 into a single linkage group, and confirmed that LG16-LG21 are also merged. The orientation and association of RH groups to each chromosome and LG was confirmed by chromosomal in situ hybridizations (FISH) of 55 BACs. Fifty RH groups were localized on the 22 chromosomes while 31 remained small orphan groups. Synteny relationships were determined between Nile tilapia, stickleback, medaka and pufferfish. CONCLUSION: The RH map and associated FISH map provide a valuable gene-ordered resource for gene mapping and QTL studies. All genetic linkage groups with their corresponding RH groups now have a corresponding chromosome which can be identified in the karyotype. Placement of conserved segments indicated that multiple inter-chromosomal rearrangements have occurred between Nile tilapia and the other model fishes. These maps represent a valuable resource for organizing the forthcoming genome sequence of Nile tilapia, and provide a foundation for evolutionary studies of East African cichlid fishes.

Genetic and physical mapping of sex-linked AFLP markers in Nile tilapia (Oreochromis niloticus).

Identification of the sex-determining genes of the Nile tilapia (Oreochromis niloticus) has important implications for commercial aquaculture. We previously identified an XX/XY sex-determining locus in this species within a 10-cM interval between markers GM201 and UNH995 on linkage group one (LG1). In order to refine this region, we developed new AFLP markers using bulked segregant analysis of the mapping families. We identified three AFLP markers that showed a sex-specific pattern of segregation. All three mapped near, but just outside, the previously identified sex-determining region on LG1. Hybridization of BAC clones containing these markers to chromosome spreads confirmed that the XX/XY sex-determining locus is on one of the small chromosomes in O. niloticus.

Use of the chromosomal co-location of the minor 5S and the major 28S rDNA as a cytogenetic marker within the genus Leuciscus (Pisces, Cyprinidae).

The chromosomes of three species from the genus Leuciscus (the ide L. idus, the European chub L. cephalus and the common dace L. leuciscus) were examined with the FISH technique for 5S and 28S rDNA probes. The analysis showed that among the three examined species, 5S rDNA signals were located on two large and four small subtelocentric chromosomes in L. leuciscus, on one large and five small subtelocentric chromosomes in L. idus, while in L. cephalus the probe signals were found on two metacentric chromosomes and one large and one small subtelocentric chromosome pairs. In all analysed species, the 28S rDNA probe signals were placed on only one chromosome pair, subtelocentric in the common dace and the European chub, and submetacentric in the ide. The three species differed in the number of sites in which both probe signals were present. In conclusion, the co-location of the 5S and 28S rDNA proved to be a useful cytogenetic marker among the studied fishes. Moreover, this marker could be adapted to other cyprinids.

Cytogenetic mapping of immunoglobulin heavy chain genes in Antarctic fish.

The chromosomal location of the IgH locus has been analyzed in several bony fish of the Antarctic perciform group Notothenioidei. Two IgH probes were prepared from the species Trematomus bernacchii (family Nototheniidae, tribe Trematominae) and mapped onto the chromosomes of ten species belonging to the same genus (Trematomus) and in two outgroups, through one-color and two-color FISH. A single location of the IgH locus was found in the majority of the species examined, including the outgroups, whereas in four of them the IgH genes splited to two chromosomal loci. RT-PCR experiments revealed the presence of three allelic sequences in T. newnesi, a species in which the IgH genes were organized in two chromosomal loci. Possible pathways leading to IgH genes duplication during the diversification of trematomine fishes were inferred from the analysis of the FISH patterns in a phylogenetic context. The present work provides the first comprehensive picture of IgH genes organization at chromosomal level in a bony fish group.

Are Cirripedia hopeful monsters? Cytogenetic approach and evidence for a Hox gene cluster in the cirripede crustacean Sacculina carcini.

The "hopeful monster" has haunted evolutionary thinking since Richard Goldschmidt coined the phrase in 1933. The phrase is directly related to genetic mechanisms in development and evolution. Cirripedes are peculiar crustaceans in that they all lack abdomens as adults. In a previous study aimed at describing the repertoire of Hox genes of the Cirripedia, we failed to isolate the abdominal-A gene in three species representative of all three cirripede orders. To address the question of whether the cirripede ancestor could have been a "hopeful monster" arising from a rearrangement of the Hox complex, we have performed a cytogenetic analysis of the Hox complex of the cirripede Sacculina carcini. We present here molecular and cytogenetic evidence for the grouping of the Hox genes on a single chromosome. This is the first direct evidence reported for the grouping of Hox genes on the same chromosome in a non-insect arthropod species.

Contrast between extensive variation of 28S rDNA and stability of 5S rDNA and telomeric repeats in the diploid-polyploid Squalius alburnoides complex and in its maternal ancestor Squalius pyrenaicus (Teleostei, Cyprinidae).

The diploid-polyploid Squalius alburnoides complex resulted from interspecific hybridization. The chromosomal mapping of 28S and 5S ribosomal genes and of (TTAGGG)n telomeric repeats was performed on specimens from the complex and from the sympatric bisexual species S. pyrenaicus (the complex maternal ancestor) as part of an investigation of the evolutionary relationships between genomic constitutions and the consequences of the ongoing polyploidization process in terms of chromosome reshaping. Contrasting results were obtained. While results with 5S rDNA and telomeric probes gave an impression of genomic stability, the variability detected with 28S rDNA probe suggested quite the opposite. The 5S rDNA probe mapped constantly to three chromosomes per haploid genome with apparently conserved locations in morphologically similar chromosomes; conversely, prominent intra- and inter-individual variations of 28S rDNA and of syntenic sites with 5S rDNA were detected with regard to number, size and location. Hypotheses for the causes of such polymorphisms are discussed. The terminal position of most 28S rDNA sites and the absence of detectable interstitial telomeric sequences suggest a mechanism that does not involve major chromosomal rearrangements. These fishes share similar patterns for the studied cytogenetic markers which may be taken as evidence of an apparent stability that may be hiding extensive and subtle genome variations that are possibly related to an ongoing evolutionary process of genome tetraploidization and speciation.

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid-tetraploid population.

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid-polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid-polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3-6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype.

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests approximately 900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

Global heterochromatic colocalization of transposable elements with minisatellites in the compact genome of the pufferfish Tetraodon nigroviridis.

Because of its unusual high degree of compaction and paucity of repetitive sequences, the genome of the smooth pufferfish Tetraodon nigroviridis is the subject of a well-advanced sequencing project. An astonishing diversity of transposable elements not found in the human and the mouse has been observed in the genome of T. nigroviridis. Due to the difficulty of assembling repeat-rich regions, the whole genome shotgun sequencing approach will probably fail to reveal the general organisation of this compact vertebrate genome. Therefore, in order to gain new insights into the global distribution pattern of repeated DNA in the genome of T. nigroviridis, we have reconstructed partial/complete repetitive sequences from data generated by the genome project and performed double-colour fluorescent in situ hybridization (FISH) analysis for representatives of three major categories of repeated sequences including two minisatellites (ms100 and ms104), two DNA transposons (Tol2 and Buffy1) and two non-long terminal repeat (LTR) retrotransposons (Rex3 and Babar). We show that DNA transposons and retroelements very frequently colocalize with minisatellites and mostly accumulate within heterochromatic regions. These results, which have not been reported so far for the fugu Takifugu rubripes, show that repeated elements are generally excluded from gene-rich regions in T. nigroviridis and underline the extreme degree of compartmentalization of this compact genome. The genome organization of the pufferfish is clearly different from that observed in humans, where repeated sequences make up an important fraction of euchromatic DNA, and is more similar to that observed in the fruit fly Drosophila melanogaster.

LINE-1 amplification accompanies explosive genome repatterning in rodents.

Transposable elements (TEs) sometimes induce karyotypic changes following recombination, breakage and rearrangement. We used FISH and Southern blot analyses to investigate the amount and distribution of LINE-1 retrotransposons in rodents (genus Taterillus, Muridae, Gerbillinae) that have recently undergone an important genome repatterning. Our results were interpreted in a known phylogenetic framework and clearly showed that LINE-1 elements were greatly amplified and non-randomly distributed in the most rearranged karyotypes. A comparison between FISH and conventional banding patterns provided evidence that LINE-1 insertion sites and chromosome breakpoints were not strongly correlated, thus suggesting that LINE-1 amplification subsequently accompanied Taterillus chromosome evolution. Similar patterns are observed in some cases of genomic stresses (hybrid genomes, cancer and DNA-damaged cells) and usually associated with DNA hypomethylation. We propose that intensively repatterned genomes face transient stress phases during which some epigenetic features, such as DNA methylation, are relaxed, thus allowing TE amplification.

An active non-LTR retrotransposon with tandem structure in the compact genome of the pufferfish Tetraodon nigroviridis.

The fish retrotransposable element Zebulon encodes a reverse transcriptase and a carboxy-terminal restriction enzyme-like endonuclease, and is related phylogenetically to site-specific non-LTR retrotransposons from nematodes. Zebulon was detected in the pufferfishes Tetraodon nigroviridis and Takifugu rubripes, as well as in the zebrafish Danio rerio. Structural analysis suggested that Zebulon, in contrast to most non-LTR retrotransposons, might be able to retrotranspose as a partial tandem array. Zebulon was active relatively recently in the compact genome of T. nigroviridis, in which it contributed to the extension of intergenic and intronic sequences, and possibly to the formation of genomic rearrangements. Accumulation of Zebulon together with other retrotransposons was observed in some heterochromatic chromosomal regions of the genome of T. nigroviridis that might serve as reservoirs for active elements. Hence, pufferfish compact genomes are not evolutionarily inert and contain active retrotransposons, suggesting the presence of mechanisms allowing accumulation of retrotransposable elements in heterochromatin, but minimizing their impact on euchromatic regions. Homologous recombination between partial tandem sequences eliminating active copies of Zebulon and reducing the size of insertions in intronic and intragenic regions might represent such a mechanism.