The late-evolving salmon and trout join the GnRH1 club.

Although it is known that the whitefish, an ancient salmonid, expresses three distinct gonadotropin-releasing hormone (GnRH) forms in the brain, it has been thought that the later-evolving salmonids (salmon and trout) had only two types of GnRH: GnRH2 and GnRH3. We now provide evidence for the expression of GnRH1 in the gonads of Atlantic salmon by rapid amplification of cDNA ends, real-time quantitative PCR and immunohistochemistry. We examined six different salmonid genomes and found that each assembly has one gene that likely encodes a viable GnRH1 prepropeptide. In contrast to both functional GnRH2 and GnRH3 paralogs, the GnRH1 homeolog can no longer express the hormone. Furthermore, the viable salmonid GnRH1 mRNA is composed of only three exons, rather than the four exons that build the GnRH2 and GnRH3 mRNAs. Transcribed gnrh1 is broadly expressed (in 17/18 tissues examined), with relative abundance highest in the ovaries. Expression of the gnrh2 and gnrh3 mRNAs is more restricted, primarily to the brain, and not in the gonads. The GnRH1 proximal promoter presents composite binding elements that predict interactions with complexes that contain diverse cell fate and differentiation transcription factors. We provide immunological evidence for GnRH1 peptide in the nucleus of 1-year-old type A spermatogonia and cortical alveoli oocytes. GnRH1 peptide was not detected during other germ cell or reproductive stages. GnRH1 activity in the salmonid gonad may occur only during early stages of development and play a key role in a regulatory network that controls mitotic and/or meiotic processes within the germ cell.

Regulatory processes that control haploid expression of salmon sperm mRNAs.

OBJECTIVE: Various stages of mRNA processing are necessary for functionally important genes required during late-stage sperm differentiation. Protein-RNA complexes form that edit, stabilize, store, deliver, localize and regulate translation of sperm mRNAs. These regulatory processes are often directed by recognition sequence elements and the particular composition of the proteins associated with the mRNAs. Previous work has shown that the cAMP response element modulator (CREM), estrogen receptor-alpha (ERα) and forkhead box L2A (FOXL2A) proteins are present in late-stage salmon sperm. Here we investigate whether these and other regulatory proteins might control processing of mRNAs not expressed until the haploid stage of development. We also examine regulatory processes that prepare and present mRNAs that generate unique products essential for differentiating sperm (i.e. for flagellar assembly and function). RESULTS: We provide evidence for potential sperm-specific recognition elements in 5'-untranslated regions (utrs) that may bind CREM, ERα, FOXL2A, Y-box and other proteins. We show that changes within the 5'-utrs and open reading frames of some sperm genes lead to distinct protein termini that may provide specific interfaces necessary for localization and function within the paternal gamete.

Subcellular localization and characterization of estrogenic pathway regulators and mediators in Atlantic salmon spermatozoal cells.

Much progress has been made regarding our understanding of aromatase regulation, estrogen synthesis partitioning and communication between the germinal and somatic compartments of the differentiating gonad. We now know that most of the enzymatic and signaling apparatus required for steroidogenesis is endogenously expressed within germ cells. However, less is known about the expression and localization of steroidogenic components within mature spermatozoa. We have assembled a sperm library presenting 197,015 putative transcripts. Co-expression clustering analysis revealed that 6687 genes were present at higher levels in sperm in comparison to fifteen other salmon tissue libraries. The sperm transcriptome is highly complex containing the highest proportion of unannotated genes (45%) of the tissues analyzed. Our analysis of highly expressed genes in late-stage sperm revealed dedication to tasks involving chromatin remodeling, flagellogenesis and proteolysis. In addition, using various different embedding and microscopic techniques, we examined the morphology of salmon spermatozoa and characterized expression and localization of several estrogenic regulatory and signaling proteins by immunohistochemistry. We provide evidence for the endogenous synthesis and localization of aromatase (CYP19A and CYP19B1) and potential mediators of estrogen [i.e., ER-alpha and soluble adenylyl cyclase (sAC)] or phosphate (i.e., CREB and FOXL2A) signaling. Partitioning of select transcripts that encode AR-beta, FSH and the LH receptor, but not AR-alpha, LH or the FSH receptor, further points to localized specificity of function in the steroidogenic circuitry of the sperm cell. These results open new avenues of investigation to further our understanding of the intra- and intercellular regulatory processes that guide sperm development and biology.

The genome and linkage map of the northern pike (Esox lucius): conserved synteny revealed between the salmonid sister group and the Neoteleostei.

The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the Atlantic salmon and rainbow trout genomes by facilitating their comparison with other major teleost groups.

Sex-specific expression and localization of aromatase and its regulators during embryonic and larval development of Atlantic salmon.

The products of dax1, foxl2a and mis have each been shown to have proliferative and/or differentiative activities during mammalian organogenesis. These factors also play a role in regulating the biosynthesis of estrogen, particularly by modulating the activity of aromatase cyp19a. We demonstrate the transcription and translation of these genes during salmon embryogenesis. We were able to track sex-specific differences in these processes through accurate determination of the sex of each embryo and larva examined from genotyped microsatellites. We detected sex- and stage-specific immunolabeling of the embryonic gut, kidney, gonads, neural cord and skeletal muscle by DAX-1, FOXL2A and MIS. These results indicate the potential of these factors to mediate proliferation and/or differentiation programs during development of these tissues. As well, immunolabeling of skeletal muscle by CYP19B1 throughout the study reveals probable neurogenic activity associated with peripheral radial glial cells and the growing embryonic musculature.

Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene.

BACKGROUND: The sablefish (order: Scorpaeniformes) is an economically important species in commercial fisheries of the North Pacific and an emerging species in aquaculture. Aside from a handful of sequences in NCBI and a few published microsatellite markers, little is known about the genetics of this species. The development of genetic tools, including polymorphic markers and a linkage map will allow for the successful development of future broodstock and mapping of phenotypes of interest. The significant sexual dimorphism between females and males makes a genetic test for early identification of sex desirable. RESULTS: A full mitochondrial genome is presented and the resulting phylogenetic analysis verifies the placement of the sablefish within the Scorpaeniformes. Nearly 35,000 assembled transcript sequences are used to identify genes and obtain polymorphic SNP and microsatellite markers. 360 transcribed polymorphic loci from two sablefish families produce a map of 24 linkage groups. The sex phenotype maps to sablefish LG14 of the male map. We show significant conserved synteny and conservation of gene-order between the threespine stickleback Gasterosteus aculeatus and sablefish. An additional 1843 polymorphic SNP markers are identified through next-generation sequencing techniques. Sex-specific markers and sequence insertions are identified immediately upstream of the gene gonadal-soma derived factor (gsdf), the master sex determinant locus in the medaka species Oryzias luzonensis. CONCLUSIONS: The first genomic resources for sablefish provide a foundation for further studies. Over 35,000 transcripts are presented, and the genetic map represents, as far as we can determine, the first linkage map for a member of the Scorpaeniformes. The observed level of conserved synteny and comparative mapping will allow the use of the stickleback genome in future genetic studies on sablefish and other related fish, particularly as a guide to whole-genome assembly. The identification of sex-specific insertions immediately upstream of a known master sex determinant implicates gsdf as an excellent candidate for the master sex determinant for sablefish.

Sex-specific expression, synthesis and localization of aromatase regulators in one-year-old Atlantic salmon ovaries and testes.

Transcripts for dax1, foxl2, mis and sf1 are co-expressed in the somatic companion cells of teleost germ cells. These regulatory factors function, in part, to modulate the transcription of aromatase, particularly cyp19a, the terminal enzyme of estrogen biosynthesis. At least two separate aromatase loci exist in teleost fish that encode distinct isoforms. The activity of two forms, cyp19a and cyp19b1, is predominantly associated with the ovary and the brain, respectively. We isolated sequences that compose the proximal promoters of cyp19a, cyp19b1 and foxl2a, to identify potential transcription factor binding motifs to define sex-specific regulatory profiles for each gene. We also provide evidence for the translation and immunological localization of DAX-1, FOXL2 and MIS to the endoplasmic reticulum and accumulation within secretory vesicles of the salmon oocyte. We found no evidence for the expression of CYP19A or CYP19B1 in the oocyte at the one-year-old stage. However, synthesis of both aromatases was localized to testicular germ and soma cells at this early stage of development. Production of these regulatory factors in the germ cells may serve to modulate the transcription and activity of endogenous aromatase and/or contribute to the differentiation of the neighbouring companion cells through secretory signaling.

Genomic resources for sea lice: analysis of ESTs and mitochondrial genomes.

Sea lice are common parasites of both farmed and wild salmon. Salmon farming constitutes an important economic market in North America, South America, and Northern Europe. Infections with sea lice can result in significant production losses. A compilation of genomic information on different genera of sea lice is an important resource for understanding their biology as well as for the study of population genetics and control strategies. We report on over 150,000 expressed sequence tags (ESTs) from five different species (Pacific Lepeophtheirus salmonis (49,672 new ESTs in addition to 14,994 previously reported ESTs), Atlantic L. salmonis (57,349 ESTs), Caligus clemensi (14,821 ESTs), Caligus rogercresseyi (32,135 ESTs), and Lernaeocera branchialis (16,441 ESTs)). For each species, ESTs were assembled into complete or partial genes and annotated by comparisons to known proteins in public databases. In addition, whole mitochondrial (mt) genome sequences of C. clemensi (13,440 bp) and C. rogercresseyi (13,468 bp) were determined and compared to L. salmonis. Both nuclear and mtDNA genes show very high levels of sequence divergence between these ectoparastic copepods suggesting that the different species of sea lice have been in existence for 37-113 million years and that parasitic association with salmonids is also quite ancient. Our ESTs and mtDNA data provide a novel resource for the study of sea louse biology, population genetics, and control strategies. This genomic information provides the material basis for the development of a 38K sea louse microarray that can be used in conjunction with our existing 44K salmon microarray to study host-parasite interactions at the molecular level. This report represents the largest genomic resource for any copepod species to date.

Regulation and expression of sexual differentiation factors in embryonic and extragonadal tissues of Atlantic salmon.

BACKGROUND: The products of cyp19, dax, foxl2, mis, sf1 and sox9 have each been associated with sex-determining processes among vertebrates. We provide evidence for expression of these regulators very early in salmonid development and in tissues outside of the hypothalamic-pituitary-adrenal/gonadal (HPAG) axis. Although the function of these factors in sexual differentiation have been defined, their roles in early development before sexual fate decisions and in tissues beyond the brain or gonad are essentially unknown. RESULTS: Bacterial artificial chromosomes containing salmon dax1 and dax2, foxl2b and mis were isolated and the regulatory regions that control their expression were characterized. Transposon integrations are implicated in the shaping of the dax and foxl2 loci. Splice variants for cyp19b1 and mis in both embryonic and adult tissues were detected and characterized. We found that cyp19b1 transcripts are generated that contain 5'-untranslated regions of different lengths due to cryptic splicing of the 3'-end of intron 1. We also demonstrate that salmon mis transcripts can encode prodomain products that present different C-termini and terminate before translation of the MIS hormone. Regulatory differences in the expression of two distinct aromatases cyp19a and cyp19b1 are exerted, despite transcription of their transactivators (ie; dax1, foxl2, sf1) occurring much earlier during embryonic development. CONCLUSIONS: We report the embryonic and extragonadal expression of dax, foxl2, mis and other differentiation factors that indicate that they have functions that are more general and not restricted to steroidogenesis and gonadogenesis. Spliced cyp19b1 and mis transcripts are generated that may provide regulatory controls for tissue- or development-specific activities. Selection of cyp19b1 transcripts may be regulated by DAX-1, FOXL2 and SF-1 complexes that bind motifs in intron 1, or by signals within exon 2 that recruit splicing factors, or both. The potential translation of proteins bearing only the N-terminal MIS prodomain may modulate the functions of other TGF ß family members in different tissues. The expression patterns of dax1 early in salmon embryogenesis implicate its role as a lineage determination factor. Other roles for these factors during embryogenesis and outside the HPAG axis are discussed.

Evolution of duplicated IgH loci in Atlantic salmon, Salmo salar.

BACKGROUND: The Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. These duplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the IGH-A (1.1 Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region have been sequenced and analyzed. RESULTS: The present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cµ region, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 VH segments which could be classified into 18 families. This is the largest number of VH families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B loci have evolved independently in the short time after the recent genome duplication approximately 60 mya. CONCLUSIONS: Our results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.

Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome.

BACKGROUND: Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. RESULTS: From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. CONCLUSIONS: 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

Regulation, expression and characterization of aromatase (cyp19b1) transcripts in ovary and testis of rainbow trout (Oncorhynchus mykiss).

Cytochrome P450 aromatase is the key enzyme in the pathway that converts androgens to estrogens. The enzyme functions in the smooth endoplasmic reticulum in a complex with NADPH-cytochrome P450 reductase. In teleost fish, at least two separate loci, cyp19a and cyp19b, encode distinct aromatase isoforms. The activity of cyp19a and cyp19b are predominantly associated with the ovary and brain, respectively, although their expression is not confined solely to these tissues. We found that at least five cyp19b1 transcripts with different 5'-UTRs are generated in the ovary and testis of rainbow trout. Regulation for selection of these variants may be through signals present in exon 2 that recruit alternative splicing factors. Also, binding elements for FOXL2 and SF-1 located within the cyp19b1 intron 1 may influence formation of transcripts that contain the 3'-end of the intron. Another transcript devoid of the exon 2 methionine initiator codon may utilize other downstream in-frame start codons. Less developed stages of ovarian and testicular tissues express only the intron-containing transcripts whereas precocious and more mature gonads express all five cyp19b1 messages. The function of these different 5'-UTRs may be for regulation of cyp19b1 at particular developmental stages or to specify control in distinct gonadal cell-types.

A salmonid EST genomic study: genes, duplications, phylogeny and microarrays.

BACKGROUND: Salmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish. RESULTS: 298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species. CONCLUSION: An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94-96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent with an ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, provide valuable new tools to study salmonids.

Isolation, characterization and comparison of Atlantic and Chinook salmon growth hormone 1 and 2.

BACKGROUND: Growth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processes in salmonids. The GH gene (gh) in salmonids is represented by duplicated, non-allelic isoforms designated as gh1 and gh2. We have isolated and characterized gh-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinook salmon (Salmo salar and Oncorhynchus tshawytscha) in order to further elucidate our understanding of the conservation and regulation of these loci. RESULTS: BACs containing gh1 and gh2 from both Atlantic and Chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genes for skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1, myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientations relative to gh1 and gh2. Viability of each of these genes was examined by PCR. We show that transposon insertions have occurred differently in the promoters of gh, within and between each species. Other differences within the promoters and intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development. CONCLUSION: A core proximal promoter for transcription of both gh1 and gh2 is conserved between the two species of salmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh1 and gh2. Additionally, organization of transposon families into the BACs containing gh1 and for the BACs containing gh2, are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the gh1- and gh2-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook and other Oncorhynchus GH1s are strikingly different in comparison to the other GHs and this change is not apparent in the surrounding non-coding sequences.

EST and mitochondrial DNA sequences support a distinct Pacific form of salmon louse, Lepeophtheirus salmonis.

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5-11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis.

Microarray analysis reveals differences in expression of cell surface and extracellular matrix components during development of the trout ovary and testis.

Trout normally spawn at 3 years of age, however, a small percentage mature a year early. This provides an opportunity to study reproductive timing and developmental processes. The ovarian and testicular extracellular matrix (ECM) participates in processes such as growth, adhesion, differentiation, cell migration and patterning. The composition of the ECM defines the interactions of specific regulatory ligands with their receptors and modulates and regulates gonadal function. To identify some of the genes involved in these processes, a 16,006-gene salmonid cDNA microarray was used to compare three-year-old normal with two-year-old normal (maturing) and with two-year-old precocious (pre-spawn) ovarian and testicular transcriptomes. We provide evidence for differences in expression of some of the genes during vasculogenesis, angiogenesis, fibrillogenesis and other processes involving ECM remodeling. Sex-specific gene expression differences of ECM components were documented between the trout ovary and testis in each developmental state. Significant differences in the expression of genes involved in translation, transcription, cell-cycling and differentiation were identified. We also report, for the first time, unequivocal evidence for the transcription of high levels of adult and embryonic hemoglobins in the developed ovary; and for the expression of transcripts that encode zona pellucida glycoproteins in both the ovary and testis of trout.

Expression of morphogenic genes in mature ovarian and testicular tissues: potential stem-cell niche markers and patterning factors.

Morphogens are developmental regulators that modulate different tissue patterning, proliferation, differentiation, or remodeling processes in embryonic and adult tissues. Morphogens may also evoke specific regulatory programs in stem cells. Some of the morphogens involved in these processes have been characterized, while others remain unidentified. A microarray containing 3,557 salmonid cDNAs was used to compare the transcriptomes of rainbow trout precocious ovary at three different stages during second year (June, August, and October) with a reference (June normal ovary) transcriptome. During this study, we detected morphogen transcript hybridizations to salmonid elements and the study was enlarged to investigate these activities in various developmental stages of both ovary and testis. Genes from diverse development regulator families such as Anterior gradient-2, BMP, Epimorphin, Flightless, Frizzled, Notch, Tiarin, Twisted gastrulation, and Wnt were demonstrated to be expressed in the adult trout gonads. In mice or rats, expression of mammalian bmp-4, epimorphin, flightless, twisted gastrulation, and GW112 transcripts were localized to cell types isolated from the developed ovary and testis. Comparisons of salmonid and mammalian morphogens at the amino acid residue level show high similarities, suggesting functional conservation. This report provides evidence for local regulation by various morphogens and their potential to control distinct programs of gene expression in the gametes and their accessory cells during gametogenesis.

Expression and genomic organization of zonadhesin-like genes in three species of fish give insight into the evolutionary history of a mosaic protein.

BACKGROUND: The mosaic sperm protein zonadhesin (ZAN) has been characterized in mammals and is implicated in species-specific egg-sperm binding interactions. The genomic structure and testes-specific expression of zonadhesin is known for many mammalian species. All zonadhesin genes characterized to date consist of meprin A5 antigen receptor tyrosine phosphatase mu (MAM) domains, mucin tandem repeats, and von Willebrand (VWD) adhesion domains. Here we investigate the genomic structure and expression of zonadhesin-like genes in three species of fish. RESULTS: The cDNA and corresponding genomic locus of a zonadhesin-like gene (zlg) in Atlantic salmon (Salmo salar) were sequenced. Zlg is similar in adhesion domain content to mammalian zonadhesin; however, the domain order is altered. Analysis of puffer fish (Takifugu rubripes) and zebrafish (Danio rerio) sequence data identified zonadhesin (zan) genes that share the same domain order, content, and a conserved syntenic relationship with mammalian zonadhesin. A zonadhesin-like gene in D. rerio was also identified. Unlike mammalian zonadhesin, D. rerio zan and S. salar zlg were expressed in the gut and not in the testes. CONCLUSION: We characterized likely orthologs of zonadhesin in both T. rubripes and D. rerio and uncovered zonadhesin-like genes in S. salar and D. rerio. Each of these genes contains MAM, mucin, and VWD domains. While these domains are associated with several proteins that show prominent gut expression, their combination is unique to zonadhesin and zonadhesin-like genes in vertebrates. The expression patterns of fish zonadhesin and zonadhesin-like genes suggest that the reproductive role of zonadhesin evolved later in the mammalian lineage.

Fish and chips: various methodologies demonstrate utility of a 16,006-gene salmonid microarray.

BACKGROUND: We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. RESULTS: We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. CONCLUSION: We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.

A physical map of the genome of Atlantic salmon, Salmo salar.

A physical map of the Atlantic salmon (Salmo salar) genome was generated based on HindIII fingerprints of a publicly available BAC (bacterial artificial chromosome) library constructed from DNA isolated from a Norwegian male. Approximately 11.5 haploid genome equivalents (185,938 clones) were successfully fingerprinted. Contigs were first assembled via FPC using high-stringency (1e-16), and then end-to-end joins yielded 4354 contigs and 37,285 singletons. The accuracy of the contig assembly was verified by hybridization and PCR analysis using genetic markers. A subset of the BACs in the library contained few or no HindIII recognition sites in their insert DNA. BglI digestion fragment patterns of these BACs allowed us to identify three classes: (1) BACs containing histone genes, (2) BACs containing rDNA-repeating units, and (3) those that do not have BglI recognition sites. End-sequence analysis of selected BACs representing these three classes confirmed the identification of the first two classes and suggested that the third class contained highly repetitive DNA corresponding to tRNAs and related sequences.