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Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.

Sex ratio depends on sex determination mechanisms and is a key demographic parameter determining population viability and resilience to natural and anthropogenic stressors. There is increasing evidence that the environment can alter sex ratio even in genetically sex-determined species (GSD), as elevated temperature can cause female-to-male sex reversal (neomales). Alarmingly, neomales are being discovered in natural populations of several fish, amphibian and reptile species worldwide. Understanding the basis of neomale development is important for conservation biology. Among GSD species, it is unknown whether those with chromosomal sex determination (CSD), the most common system, will better resist the influence of high temperature than those with polygenic sex determination (PSD). Here, we compared the effects of elevated temperature in two wild zebrafish strains, Nadia (NA) and Ekkwill (EKW), which have CSD with a ZZ/ZW system, against the AB laboratory strain, which has PSD. First, we uncovered novel sex genotypes and the results showed that, at control temperature, the masculinization rate roughly doubled with the addition of each Z chromosome, while some ZW and WW fish of the wild strains became neomales. Surprisingly, we found that at elevated temperatures WW fish were just as likely as ZW fish to become neomales and that all strains were equally susceptible to masculinization. These results demonstrate that the Z chromosome is not essential for male development and that the dose of W buffers masculinization at the control temperature but not at elevated temperature. Furthermore, at the elevated temperature the testes of neomales, but not of normal males, contained more spermatozoa than at the control temperature. Our results show in an unprecedented way that, in a global warming scenario, CSD species may not necessarily be better protected against the masculinizing effect of elevated temperature than PSD species, and reveal genotype-by-temperature interaction

Climate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystems

PurposeGrowth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants.MethodsWe present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality.ResultsPatients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants.ConclusionGDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.

Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes’ roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

PurposeGrowth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants.MethodsWe present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality.ResultsPatients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants.ConclusionGDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

Sexual selection results in sex-specific characters like the conspicuously pigmented extension of the ventral tip of the caudal fin-the ``sword''-in males of several species of Xiphophorus fishes. To uncover the genetic architecture underlying sword formation and to identify genes that are associated with its development, we characterized the sword transcriptional profile and combined it with genetic mapping approaches. Results showed that the male ornament of swordtails develops from a sexually non-dimorphic prepattern of transcription factors in the caudal fin. Among genes that constitute the exclusive sword transcriptome and are located in the genomic region associated with this trait we identify the potassium channel, Kcnh8, as a sword development gene. In addition to its neural function kcnh8 performs a known role in fin growth. These findings indicate that during evolution of swordtails a brain gene has been co-opted for an additional novel function in establishing a male ornament.

Background Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades. Results We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia. Conclusions The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and T

Background Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades. Results We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia. Conclusions The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and T

Sex determination in fish is a labile trait easily influenced by environmental factors, often temperature. A mismatch between genotypic and phenotypic sex usually tends towards genotypic females developing as males referred to as neomales. Neomales are being discovered in wild populations of several fish species. Thus, understanding the genetic basis leading toward neomales is of great interest in a global climate change scenario. Wild zebrafish strains possess a ZZ/ZW chromosomal sex determination system, in contrast to laboratory strains, which lack a Z chromosome and assume a polygenic sex determination mechanism. Whether temperature alters the sex ratio in wild strains resulting in neomales and whether the gonadal transcriptome differs between genetic males (ZZm) and neomales (ZWm) is unknown. We assessed the rate of sex reversal in Nadia and EkkWill wild strains using different families exposed to 28 °C (control) and 34¿36 °C (masculinizing) temperatures during sex. In adults, histomorphometrics and transcriptomic analyses by RNA sequencing provided insights into gonadal morphology and function. Results showed, surprisingly, that both wild strains were at least as susceptible to the masculinizing effects of elevated temperature as the laboratory strain, with abundant spontaneous and temperature-induced neomales, although the two natural strains had different genotype-by-environment interactions. Histologically, Nadia ZWm but not ZZm showed a higher number of spermatozoa after exposure to elevated temperature. Transcriptomic results had a strong family influence. Further, ZWm testes had a transcriptome profile indistinguishable from ZZm testes. Taken together, these results suggest a genetic basis underlying both the production of neomales and their response to temperature with potential functional consequences in reproductive capacity. Identifying the genes involved will improve our understanding of the mechanisms of zebrafish sex determination

Motivation: MicroRNAs (miRNAs) are small RNA molecules (similar to 22 nucleotide long) involved in post-transcriptional gene regulation. Advances in high-throughput sequencing technologies led to the discovery of isomiRs, which are miRNA sequence variants. While many miRNA-seq analysis tools exist, the diversity of output formats hinders accurate comparisons between tools and precludes data sharing and the development of common downstream analysis methods. Results: To overcome this situation, we present here a community-based project, miRNA Transcriptomic Open Project (miRTOP) working towards the optimization of miRNA analyses. The aim of miRTOP is to promote the development of downstream isomiR analysis tools that are compatible with existing detection and quantification tools. Based on the existing GFF3 format, we first created a new standard format, mirGFF3, for the output of miRNA/isomiR detection and quantification results from small RNA-seq data. Additionally, we developed a command line Python tool, mirtop, to create and manage the mirGFF3 format. Currently, mirtop can convert into mirGFF3 the outputs of commonly used pipelines, such as seqbuster, isomiR-SEA, sRNAbench, Prost! as well as BAM files. Some tools have also incorporated the mirGFF3 format directly into their code, such as, miRge2.0, IsoMIRmap and OptimiR. Its open architecture enables any tool or pipeline to output or convert results into mirGFF3. Collectively, this isomiR categorization system, along with the accompanying mirGFF3 and mirtop API, provide a comprehensive solution for the standardization of miRNA and isomiR annotation, enabling data sharing, reporting, comparative analyses and benchmarking, while promoting the development of common miRNA methods focusing on downstream steps of miRNA detection, annotation and quantification.

Background Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades. Results We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia. Conclusions The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and T

Background Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades. Results We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia. Conclusions The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and T

Sex determination in fish is a labile trait easily influenced by environmental factors, often temperature. A mismatch between genotypic and phenotypic sex usually tends towards genotypic females developing as males referred to as neomales. Neomales are being discovered in wild populations of several fish species. Thus, understanding the genetic basis leading toward neomales is of great interest in a global climate change scenario. Wild zebrafish strains possess a ZZ/ZW chromosomal sex determination system, in contrast to laboratory strains, which lack a Z chromosome and assume a polygenic sex determination mechanism. Whether temperature alters the sex ratio in wild strains resulting in neomales and whether the gonadal transcriptome differs between genetic males (ZZm) and neomales (ZWm) is unknown. We assessed the rate of sex reversal in Nadia and EkkWill wild strains using different families exposed to 28 °C (control) and 34¿36 °C (masculinizing) temperatures during sex. In adults, histomorphometrics and transcriptomic analyses by RNA sequencing provided insights into gonadal morphology and function. Results showed, surprisingly, that both wild strains were at least as susceptible to the masculinizing effects of elevated temperature as the laboratory strain, with abundant spontaneous and temperature-induced neomales, although the two natural strains had different genotype-by-environment interactions. Histologically, Nadia ZWm but not ZZm showed a higher number of spermatozoa after exposure to elevated temperature. Transcriptomic results had a strong family influence. Further, ZWm testes had a transcriptome profile indistinguishable from ZZm testes. Taken together, these results suggest a genetic basis underlying both the production of neomales and their response to temperature with potential functional consequences in reproductive capacity. Identifying the genes involved will improve our understanding of the mechanisms of zebrafish sex determination

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution., Group Authors: Undiagnosed Dis Network ; Care4Rare Canada Consortium

In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as males or females. Many sex determination and gonad differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Müllerian duct development, where it antagonizes the male-promoting FGF9 signal. Wnt4 is well conserved across all vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs in nonmammalian vertebrates. This question is especially interesting in teleosts, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4, and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination and/or differentiation in zebrafish. Additionally, both male and female wnt4a mutants are sterile due to defects in reproductive duct development. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in mammalian and nonmammalian vertebrates.

Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes.

The evolution of antifreeze glycoproteins has enabled notothenioid fish to flourish in the freezing waters of the Southern Ocean.Whereas successful at the biodiversity level to life in the cold, paradoxically at the cellular level these stenothermal animals have problems producing, folding, and degrading proteins at their ambient temperatures of –1.86 °C. In this first multi-species transcriptome comparison of the amino acid composition of notothenioid proteinswith temperate teleostproteins,we show that, unlike psychrophilic bacteria, Antarctic fish provide little evidence for the mass alteration of protein amino acid composition to enhance protein folding and reduce protein denaturation in the cold. The exception was the significant overrepresentation of positions where leucine in temperate fish proteins was replaced by methionine in the notothenioid orthologues. We hypothesize that these extra methionines have been preferentially assimilated into the genome to act as redox sensors in the highly oxygenated waters of the Southern Ocean. This redox hypothesis is supported by analyses of notothenioids showing enrichment of genes associated with responses to environmental stress, particularly reactive oxygen species. So overall, although notothenioid fish show cold-associated problems with protein homeostasis, they may have modified only a selected number of biochemical pathways to work efficiently below 0°C. Even a slight warming of the Southern Ocean might disrupt the critical functions of this handful of key pathways with considerable impacts for the functioning of this ecosystem in the future.

In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as males or females. Many sex determination and gonad differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Müllerian duct development, where it antagonizes the male-promoting FGF9 signal. Wnt4 is well conserved across all vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs in nonmammalian vertebrates. This question is especially interesting in teleosts, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4, and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination and/or differentiation in zebrafish. Additionally, both male and female wnt4a mutants are sterile due to defects in reproductive duct development. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in mammalian and nonmammalian vertebrates.

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.

Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes.

The evolution of antifreeze glycoproteins has enabled notothenioid fish to flourish in the freezing waters of the Southern Ocean.Whereas successful at the biodiversity level to life in the cold, paradoxically at the cellular level these stenothermal animals have problems producing, folding, and degrading proteins at their ambient temperatures of –1.86 °C. In this first multi-species transcriptome comparison of the amino acid composition of notothenioid proteinswith temperate teleostproteins,we show that, unlike psychrophilic bacteria, Antarctic fish provide little evidence for the mass alteration of protein amino acid composition to enhance protein folding and reduce protein denaturation in the cold. The exception was the significant overrepresentation of positions where leucine in temperate fish proteins was replaced by methionine in the notothenioid orthologues. We hypothesize that these extra methionines have been preferentially assimilated into the genome to act as redox sensors in the highly oxygenated waters of the Southern Ocean. This redox hypothesis is supported by analyses of notothenioids showing enrichment of genes associated with responses to environmental stress, particularly reactive oxygen species. So overall, although notothenioid fish show cold-associated problems with protein homeostasis, they may have modified only a selected number of biochemical pathways to work efficiently below 0°C. Even a slight warming of the Southern Ocean might disrupt the critical functions of this handful of key pathways with considerable impacts for the functioning of this ecosystem in the future.

BackgroundMany patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added.MethodsWe evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care.ResultsA total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes.ConclusionsThe UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.).

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti‐signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black–brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray‐finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral‐graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray‐finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences., Funding: We thank the Broad Institute Genomics Platform for constructing and sequencing gar DNA and RNA libraries and J. Turner-Maier for the gar transcriptome assembly. We thank the teams of the Bayousphere Research Laboratory (Nicholls State University) and the University of Oregon Fish Facility for gar work and husbandry. We thank J. Westlund for the design of the species illustrations. The generation of gar sequences and assemblies by the Broad Institute of MIT and Harvard University was supported by US National Institutes of Health (NIH)/National Human Genome Research Institute grant U54 HG03067. This work was further supported by US NIH grants R01 OD011116 (alias R01 RR020833) and R24 OD01119004 (J.H.P.); a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation and the Volkswagen Foundation Initiative Evolutionary Biology, grant I/84 815 (I.B.); US NIH grant T32 HD055164 and National Science Foundation (NSF) Doctoral Dissertation Improvement Grant 1311436 (A.R.G.); Uehara Memorial Foundation Research Fellowship 2013, Japan Society for the Promotion of Science Postdoctoral Research Fellowship 2012-127 and Marine Biological Laboratory Research Award 2014 (T.N.); Brazilian National Council for Scientific and Technological Development (CNPq) grants 402754/2012-3 and 477658/2012-1 (I.S.); the Brinson Foundation and the University of Chicago Biological Sciences Division (N.H.S.); NSF grant BCS0725227 (K.K.); call 'ARISTEIA I' of the National Strategic Reference Framework 2007-2013 (SPARCOMP, 36), Ministry of Education and Religious Affairs of Greece (T.M.); Agence Nationale de la Recherche (ANR) grant ANR-10-GENM-017 (PhyloFish; J.B.); the Wellcome Trust (grants WT095908 and WT098051) and the European Molecular Biology Laboratory (D.B., S.M.J.S. and B.A.); the Biomedical Research Council of the Agency for Science, Technology and Research (A*STAR), Singapore (B.V.); European Research Council grant 268513 (P.W.H.H. and K.J.M.); the Ministerio de Ciencia

Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome.

Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this "RAD-sex" method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, "male genotypes" became males and some "female genotypes" also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature 496 (2013): 311-316, doi:10.1038/nature12027., The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution., cquisition and storage of Latimeria chalumnae samples was supported by grants from the African Coelacanth Ecosystem Programme of the South African National Department of Science and Technology. Generation of the Latimeria chalumnae and Protopterus annectens sequences by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was supported by grants from the National Human Genome Research Institute (NHGRI). K.L.T. is the recipient of a EURYI award from the European Science Foundation.

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature 496 (2013): 311-316, doi:10.1038/nature12027., The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution., cquisition and storage of Latimeria chalumnae samples was supported by grants from the African Coelacanth Ecosystem Programme of the South African National Department of Science and Technology. Generation of the Latimeria chalumnae and Protopterus annectens sequences by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was supported by grants from the National Human Genome Research Institute (NHGRI). K.L.T. is the recipient of a EURYI award from the European Science Foundation.

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca(2+)-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers., Kathy and Curt Marble Cancer Research Fund, Arthur C. Merrill, National Institutes of Health (U.S.) (Grant CA106416), National Institutes of Health (U.S.) (Grant ROI RR020833), National Institutes of Health (U.S.) (Grant 1F32GM095213-01)

Within vertebrates, major sex determining genes can differ among taxa and even within species. In zebrafish (Danio rerio), neither heteromorphic sex chromosomes nor single sex determination genes of large effect, like Sry in mammals, have yet been identified. Furthermore, environmental factors can influence zebrafish sex determination. Although progress has been made in understanding zebrafish gonad differentiation (e.g. the influence of germ cells on gonad fate), the primary genetic basis of zebrafish sex determination remains poorly understood. To identify genetic loci associated with sex, we analyzed F(2) offspring of reciprocal crosses between Oregon *AB and Nadia (NA) wild-type zebrafish stocks. Genome-wide linkage analysis, using more than 5,000 sequence-based polymorphic restriction site associated (RAD-tag) markers and population genomic analysis of more than 30,000 single nucleotide polymorphisms in our *ABxNA crosses revealed a sex-associated locus on the end of the long arm of chr-4 for both cross families, and an additional locus in the middle of chr-3 in one cross family. Additional sequencing showed that two SNPs in dmrt1 previously suggested to be functional candidates for sex determination in a cross of ABxIndia wild-type zebrafish, are not associated with sex in our AB fish. Our data show that sex determination in zebrafish is polygenic and that different genes may influence sex determination in different strains or that different genes become more important under different environmental conditions. The association of the end of chr-4 with sex is remarkable because, unique in the karyotype, this chromosome arm shares features with known sex chromosomes: it is highly heterochromatic, repetitive, late replicating, and has reduced recombination. Our results reveal that chr-4 has functional and structural properties expected of a sex chromosome.

Sparc (Osteonectin), a matricellular glycoprotein expressed by many differentiated cells, is a major non-collagenous constituent of vertebrate bones. recent studies indicate that Sparc expression appears early in development, although its function and regulation during embryogenesis are largely unknown. We cloned zebrafish sparc and investigated its role during development, using a morpholino antisense oligonucleotide-based knockdown approach. Knockdown of Sparc function resulted in specific skeletal, vestibular and haematopoietic defects that are highlighted by changes in gene expression, morphology and behavior. We rescued the knockdown phenotypes by co-injecting sparc mrNA, providing evidence that the knockdown phenotype is due specifically to impairment of Sparc function. We conclude that the matricellular protein osteoenectin is required for bone formation, vestibular development and normal differentiation of erythoid and myeloid lineage cells

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.