Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Reconstruction of proto-vertebrate, proto-cyclostome and proto-gnathostome genomes provides new insights into early vertebrate evolution.

Ancient polyploidization events have had a lasting impact on vertebrate genome structure, organization and function. Some key questions regarding the number of ancient polyploidization events and their timing in relation to the cyclostome-gnathostome divergence have remained contentious. Here we generate de novo long-read-based chromosome-scale genome assemblies for the Japanese lamprey and elephant shark. Using these and other representative genomes and developing algorithms for the probabilistic macrosynteny model, we reconstruct high-resolution proto-vertebrate, proto-cyclostome and proto-gnathostome genomes. Our reconstructions resolve key questions regarding the early evolutionary history of vertebrates. First, cyclostomes diverged from the lineage leading to gnathostomes after a shared tetraploidization (1R) but before a gnathostome-specific tetraploidization (2R). Second, the cyclostome lineage experienced an additional hexaploidization. Third, 2R in the gnathostome lineage was an allotetraploidization event, and biased gene loss from one of the subgenomes shaped the gnathostome genome by giving rise to remarkably conserved microchromosomes. Thus, our reconstructions reveal the major evolutionary events and offer new insights into the origin and evolution of vertebrate genomes.

Antigen receptor repertoires of one of the smallest known vertebrates.

The rules underlying the structure of antigen receptor repertoires are not yet fully defined, despite their enormous importance for the understanding of adaptive immunity. With current technology, the large antigen receptor repertoires of mice and humans cannot be comprehensively studied. To circumvent the problems associated with incomplete sampling, we have studied the immunogenetic features of one of the smallest known vertebrates, the cyprinid fish Paedocypris sp. "Singkep" ("minifish"). Despite its small size, minifish has the key genetic facilities characterizing the principal vertebrate lymphocyte lineages. As described for mammals, the frequency distributions of immunoglobulin and T cell receptor clonotypes exhibit the features of fractal systems, demonstrating that self-similarity is a fundamental property of antigen receptor repertoires of vertebrates, irrespective of body size. Hence, minifish achieve immunocompetence via a few thousand lymphocytes organized in robust scale-free networks, thereby ensuring immune reactivity even when cells are lost or clone sizes fluctuate during immune responses.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Conservation as well as divergence in Mcidas function underlies the differentiation of multiciliated cells in vertebrates.

Multiciliated cells (MCCs) differentiate hundreds of motile cilia that beat to drive fluid movement over various kinds of epithelia. In Xenopus, mice and human, the coiled-coil containing protein Mcidas (Mci) has been shown to be a key transcriptional regulator of MCC differentiation. We have examined Mci function in the zebrafish, another model organism that is widely used to study ciliary biology. We show that zebrafish mci is expressed specifically in the developing MCCs of the kidney tubules, but surprisingly, not in those of the nasal placodes. Mci proteins lack a DNA binding domain and associate with the cell-cycle transcription factors E2f4/5 for regulating MCC-specific gene expression. We found that while the zebrafish Mci protein can complex with the E2f family members, its sequence as well as the requirement and sufficiency for MCC differentiation has diverged significantly from Mci homologues of the tetrapods. We also provide evidence that compared to Gmnc, another related coiled-coil protein that has recently been shown to regulate MCC development upstream of Mci, the Mci protein originated later within the vertebrate lineage. Based on these data, we argue that in contrast to Gmnc, which has a vital role in the genetic circuitry that drives MCC formation, the requirement of Mci, at least in the zebrafish, is not obligatory.

Chromosome-level genome assembly of the coastal horseshoe crab (Tachypleus gigas).

Horseshoe crabs, represented by only four extant species, have existed for around 500 million years. However, their existence is now under threat because of anthropogenic activities. The availability of genomic resources for these species will be valuable in planning appropriate conservation measures. Whole-genome sequences are currently available for three species. In this study, we have generated a chromosome-level genome assembly of the fourth species, the Asian coastal horseshoe crab (Tachypleus gigas; genome size 2.0 Gb). The genome assembly has a scaffold N50 value of 140 Mb with ~97% of the assembly mapped to 14 scaffolds representing 14 chromosomes of T. gigas. In addition, we have generated the complete mitochondrial genome sequence and deep-coverage transcriptome assemblies for four tissues. A total of 26,159 protein-coding genes were predicted in the genome. The T. gigas genome contains five Hox clusters similar to the mangrove horseshoe crab (Carcinoscorpius rotundicauda), suggesting that the common ancestor of horseshoe crabs already possessed five Hox clusters. Phylogenomic and divergence time analysis suggested that the American and Asian horseshoe crab lineages shared a common ancestor around the Silurian period (~436 Ma). Comparison of the T. gigas genome with those of other horseshoe crab species with chromosome-level assemblies provided insights into the chromosomal rearrangement events that occurred during the emergence of these species. The genomic resources of T. gigas will be useful for understanding their genetic diversity and population structure and would help in designing strategies for managing and conserving their stocks across Asia.

Chromosome-level assembly of the horseshoe crab genome provides insights into its genome evolution.

The evolutionary history of horseshoe crabs, spanning approximately 500 million years, is characterized by remarkable morphological stasis and a low species diversity with only four extant species. Here we report a chromosome-level genome assembly for the mangrove horseshoe crab (Carcinoscorpius rotundicauda) using PacBio reads and Hi-C data. The assembly spans 1.67 Gb with contig N50 of 7.8 Mb and 98% of the genome assigned to 16 chromosomes. The genome contains five Hox clusters with 34 Hox genes, the highest number reported in any invertebrate. Detailed analysis of the genome provides evidence that suggests three rounds of whole-genome duplication (WGD), raising questions about the relationship between WGD and species radiation. Several gene families, particularly those involved in innate immunity, have undergone extensive tandem duplication. These expanded gene families may be important components of the innate immune system of horseshoe crabs, whose amebocyte lysate is a sensitive agent for detecting endotoxin contamination.

Comparative genomics reveal shared genomic changes in syngnathid fishes and signatures of genetic convergence with placental mammals.

Syngnathids (seahorses, pipefishes and seadragons) exhibit an array of morphological innovations including loss of pelvic fins, a toothless tubular mouth and male pregnancy. They comprise two subfamilies: Syngnathinae and Nerophinae. Genomes of three Syngnathinae members have been analyzed previously. In this study, we have sequenced the genome of a Nerophinae member, the Manado pipefish (Microphis manadensis), which has a semi-enclosed brood pouch. Comparative genomic analysis revealed that the molecular evolutionary rate of the four syngnathids is higher than that of other teleosts. The loss of all but one P/Q-rich SCPP gene in the syngnathids suggests a role for the lost genes in dentin and enameloid formation in teleosts. Genome-wide comparison identified a set of 118 genes with parallel identical amino acid substitutions in syngnathids and placental mammals. Association of some of these genes with placental and embryonic development in mammals suggests a role for them in syngnathid pregnancy.

Lampreys, the jawless vertebrates, contain three Pax6 genes with distinct expression in eye, brain and pancreas.

The transcription factor Pax6 is crucial for the development of the central nervous system, eye, olfactory system and pancreas, and is implicated in human disease. While a single Pax6 gene exists in human and chicken, Pax6 occurs as a gene family in other vertebrates, with two members in elephant shark, Xenopus tropicalis and Anolis lizard and three members in teleost fish such as stickleback and medaka. However, the complement of Pax6 genes in jawless vertebrates (cyclostomes), the sister group of jawed vertebrates (gnathostomes), is unknown. Using a combination of BAC sequencing and genome analysis, we discovered three Pax6 genes in lampreys. Unlike the paired-less Pax6 present in some gnathostomes, all three lamprey Pax6 have a highly conserved full-length paired domain. All three Pax6 genes are expressed in the eye and brain, with variable expression in other tissues. Notably, lamprey Pax6α transcripts are found in the pancreas, a vertebrate-specific organ, indicating the involvement of Pax6 in development of the pancreas in the vertebrate ancestor. Multi-species sequence comparisons revealed only a single conserved non-coding element, in the lamprey Pax6ß locus, with similarity to the PAX6 neuroretina enhancer. Using a transgenic zebrafish enhancer assay we demonstrate functional conservation of this element over 500 million years of vertebrate evolution.

The Divergent Genomes of Teleosts.

Boasting nearly 30,000 species, teleosts account for half of all extant vertebrates and approximately 98% of all ray-finned fish species (Actinopterygii). Teleosts are also the largest and most diverse group of vertebrates, exhibiting an astonishing level of morphological, physiological, and behavioral diversity. Previous studies had indicated that the teleost lineage has experienced an additional whole-genome duplication event. Recent comparative genomic analyses of teleosts and other bony vertebrates using spotted gar (a nonteleost ray-finned fish) and elephant shark (a cartilaginous fish) as outgroups have revealed several divergent features of teleost genomes. These include an accelerated evolutionary rate of protein-coding and nucleotide sequences, a higher rate of intron turnover, loss of many potential cis-regulatory elements and shorter conserved syntenic blocks. A combination of these divergent genomic features might have contributed to the evolution of the amazing phenotypic diversity and morphological innovations of teleosts.

Lampreys, the jawless vertebrates, contain only two ParaHox gene clusters.

ParaHox genes (Gsx, Pdx, and Cdx) are an ancient family of developmental genes closely related to the Hox genes. They play critical roles in the patterning of brain and gut. The basal chordate, amphioxus, contains a single ParaHox cluster comprising one member of each family, whereas nonteleost jawed vertebrates contain four ParaHox genomic loci with six or seven ParaHox genes. Teleosts, which have experienced an additional whole-genome duplication, contain six ParaHox genomic loci with six ParaHox genes. Jawless vertebrates, represented by lampreys and hagfish, are the most ancient group of vertebrates and are crucial for understanding the origin and evolution of vertebrate gene families. We have previously shown that lampreys contain six Hox gene loci. Here we report that lampreys contain only two ParaHox gene clusters (designated as α- and ß-clusters) bearing five ParaHox genes (Gsxα, Pdxα, Cdxα, Gsxß, and Cdxß). The order and orientation of the three genes in the α-cluster are identical to that of the single cluster in amphioxus. However, the orientation of Gsxß in the ß-cluster is inverted. Interestingly, Gsxß is expressed in the eye, unlike its homologs in jawed vertebrates, which are expressed mainly in the brain. The lamprey Pdxα is expressed in the pancreas similar to jawed vertebrate Pdx genes, indicating that the pancreatic expression of Pdx was acquired before the divergence of jawless and jawed vertebrate lineages. It is likely that the lamprey Pdxα plays a crucial role in pancreas specification and insulin production similar to the Pdx of jawed vertebrates.

A chromosome-level genome assembly of the Asian arowana, Scleropages formosus.

Asian arowana (Scleropages formosus), an ancient teleost belonging to the Order Osteoglossomorpha, has been a valuable ornamental fish with some varieties. However, its biological studies and breeding germplasm have been remarkably limited by the lack of a reference genome. To solve these problems, here we report high-quality genome sequences of three common varieties of Asian arowana (the golden, red and green arowana). We firstly generated a chromosome-level genome assembly of the golden arowana, on basis of the genetic linkage map constructed with the restriction site-associated DNA sequencing (RAD-seq). In addition, we obtained draft genome assemblies of the red and green varieties. Finally, we annotated 22,016, 21,256 and 21,524 protein-coding genes in the genome assemblies of golden, red and green varieties respectively. Our data were deposited in publicly accessible repositories to promote biological research and molecular breeding of Asian arowana.

The seahorse genome and the evolution of its specialized morphology.

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

The genome of the largest bony fish, ocean sunfish (Mola mola), provides insights into its fast growth rate.

BACKGROUND: The ocean sunfish (Mola mola), which can grow up to a length of 2.7 m and weigh 2.3 tons, is the world's largest bony fish. It has an extremely fast growth rate and its endoskeleton is mainly composed of cartilage. Another unique feature of the sunfish is its lack of a caudal fin, which is replaced by a broad and stiff lobe that results in the characteristic truncated appearance of the fish. RESULTS: To gain insights into the genomic basis of these phenotypic traits, we sequenced the sunfish genome and performed a comparative analysis with other teleost genomes. Several sunfish genes involved in the growth hormone and insulin-like growth factor 1 (GH/IGF1) axis signalling pathway were found to be under positive selection or accelerated evolution, which might explain its fast growth rate and large body size. A number of genes associated with the extracellular matrix, some of which are involved in the regulation of bone and cartilage development, have also undergone positive selection or accelerated evolution. A comparison of the sunfish genome with that of the pufferfish (fugu), which has a caudal fin, revealed that the sunfish contains more homeobox (Hox) genes although both genomes contain seven Hox clusters. Thus, caudal fin loss in sunfish is not associated with the loss of a specific Hox gene. CONCLUSIONS: Our analyses provide insights into the molecular basis of the fast growth rate and large size of the ocean sunfish. The high-quality genome assembly generated in this study should facilitate further studies of this 'natural mutant'.

Identification of three somatostatin genes in lampreys.

Somatostatins (SSs) are a structurally diverse family of neuropeptides that play important roles in the regulation of growth, development and metabolism in vertebrates. It has been recently proposed that the common ancestor of gnathostomes possessed three SS genes, namely SS1, SS2 and SS5. SS1 and SS2 are still present in most extant gnathostome species investigated so far while SS5 primarily occurs in chondrichthyes, actinopterygians and actinistia but not in tetrapods. Very little is known about the repertoire of SSs in cyclostomes, which are extant jawless vertebrates. In the present study, we report the cloning of the cDNAs encoding three distinct lamprey SS variants that we call SSa, SSb and SSc. SSa and SSb correspond to the two SS variants previously characterized in lamprey, while SSc appears to be a totally novel one. SSa exhibits the same sequence as gnathostome SS1. SSb differs from SSa by only one substitution (Thr12→Ser). SSc exhibits a totally unique structure (ANCRMFYWKTMAAC) that shares only 50% identity with SSa and SSb. SSa, SSb and SSc precursors do not exhibit any appreciable sequence similarity outside the C-terminal region containing the SS sequence. Phylogenetic analyses failed to clearly assign orthology relationships between lamprey and gnathostome SS genes. Synteny analysis suggests that the SSc gene arose before the split of the three gnathostome genes SS1, SS2 and SS5.