Survey sequencing and comparative analysis of the elephant shark (Callorhinchus milii) genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Glial architecture of the ghost shark (Callorhinchus milii, Holocephali, Chondrichthyes) as revealed by different immunohistochemical markers

This article presents the first study on the glial architecture of a representative species of Holocephali, Callorhinchus milii (ghost shark). Holocephali are a small subclass of Chondrichthyes, with only a few extant genera, and those are considered to have a brain organization more similar to squalomorph sharks than to galeomorph sharks, skates, and rays. Three different astroglial markers--glial fibrillary acidic protein, S-100 protein, and glutamine synthetase (GS)--were investigated by immunohistochemical methods, applying both diaminobenzidine (DAB) and fluorescent techniques. They revealed similar glial structures, although most of them were detected by immunohistochemical reaction against GS and visualized by DAB. The predominant elements were radial ependymoglia spanning the area between the ventricular and meningeal surfaces, as in squalomorph sharks. Other similar features were the light appearance of myelinated neural tracts devoid of immunoreactivity, and the glial architecture of the reticular formation of the brain stem, cerebellum, and tectum, the latter with recognizable layers. The immunoreactivity of the vascular walls was similar; however, it is believed that different cell types form the blood-brain barrier in chimeras and in elasmobranchs. Some glial structures, however, resembled those of skates, rays, and galeomorph sharks. In C. milii astrocyte-like elements were observed in the telencephalon, using GS and S-100, although typical astrocyte-rich regions were not found. In some areas, especially the telencephalon, not only endfeet but also cell bodies were observed to be attached to the meningeal surface, with processes extending into the brain substance.     

Embryonic staging and external features of development of the Chimaeroid fish,Callorhinchus milii (Holocephali,Callorhinchidae)

The development of Callorhinchus milii, a primitive chondrichthyan fish (Subclass Holocephali) is described in detail based on a complete series of embryos from stage 17 to hatching. The external features of these specimens, in comparison with other chondrichthyan embryos, are used to establish the first staging table for any chimaeroid species. Each stage of C. milii is defined by a suite of morphological characters in addition to total length, including the number of somites, extent of external pigmentation, eye size and shape, head flexure, heart morphology, and size and shape of paired and unpaired fins. Particular attention is given to features of the gill arches and associated structures, including external gill filaments and the opercular flap. Embryos of this species also possess a transient rostral bulb, a feature unique to chimaeroids. Embryological development of Callorhinchus milii is similar to that previously described for sharks and batoids (Subclass Elasmobranchii), including the spiny dogfish, Squalus acanthias, the Japanese bullshark, Heterodontus japonicus, the lesser spotted dogfish, Scyliorhinus canicula, the frill shark, Chlamydoselachus anguineus, the guitarfish, Rhinobatus halavi, and the skate, Raja brachyura. Callorhinchus milii is also similar in overall development to another holocephalan, Hydrolagus colliei. A review of previous staging schemes confirms that early morphological development in all three major chondrichthyan lineages (sharks, batoids, and chimaeras) can be correlated using a common set of stages. A uniform staging system is provided that should prove useful in continuing ontogenetic and phylogenetic studies of this entire clade of fishes. J. Morphol. 236:25–47, 1998. © 1998 Wiley-Liss, Inc.     

Evolution and Functional Characterisation of Melanopsins in a Deep-Sea Chimaera (Elephant Shark,Callorhinchus milii)

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.     

Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region

The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a K(d) of 0.80 +/- 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a K(d) of 0.14 +/- 0.01 nm (mean +/- SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.     

Functional conservation of a forebrain enhancer from the elephant shark (<Emphasis Type="Italic">Callorhinchus milii</Emphasis>) in zebrafish and mice

Background  

The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse.     

Characterization of the neurohypophysial hormone gene loci in elephant shark and the Japanese lamprey: origin of the vertebrate neurohypophysial hormone genes

Background  

Vasopressin and oxytocin are mammalian neurohypophysial hormones with distinct functions. Vasopressin is involved mainly in osmoregulation and oxytocin is involved primarily in parturition and lactation. Jawed vertebrates contain at least one homolog each of vasopressin and oxytocin, whereas only a vasopressin-family hormone, vasotocin, has been identified in jawless vertebrates. The genes encoding vasopressin and oxytocin are closely linked tail-to-tail in eutherian mammals whereas their homologs in chicken, Xenopus and coelacanth (vasotocin and mesotocin) are linked tail-to-head. In contrast, their pufferfish homologs, vasotocin and isotocin, are located on the same strand of DNA with isotocin located upstream of vasotocin and separated by five genes. These differences in the arrangement of the two genes in different bony vertebrate lineages raise questions about their origin and ancestral arrangement. To trace the origin of these genes, we have sequenced BAC clones from the neurohypophysial gene loci in a cartilaginous fish, the elephant shark (Callorhinchus milii), and in a jawless vertebrate, the Japanese lamprey (Lethenteron japonicum). We have also analyzed the neurohypophysial hormone gene locus in an invertebrate chordate, the amphioxus (Branchiostoma floridae).     

Recent segmental and gene duplications in the mouse genome

Background

The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (≥ 5 kb) and recent (≥ 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies.

Results

We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice.

Conclusion

Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the identified duplication content presented in this work. This data will also be relevant to the growing number of investigators who use the draft genome sequence for experimental design and analysis.

     

Vasoactive receptors in abdominal blood vessels of the dogfish shark, Squalus acanthias

Previous studies have demonstrated that the ventral aorta of the dogfish shark, Squalus acanthias, responds to a variety of cell-signaling agents. To investigate the generality of vasoactive receptors in the shark vasculature, in particular a conductance artery (anterior mesenteric) and vein (posterior intestinal), I measured the effect of acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostaglandins on tension in isolated rings from these vessels. Both vessels responded to these agents, and responses to receptor-specific ligands for endothelin and natriuretic peptide receptors suggest that B-type endothelin receptors are expressed in both vessels and that the artery expresses both A- and B-type natriuretic peptide receptors; however, the vein (like the ventral aorta) expresses only the B-type natriuretic peptide receptor. My data suggest that a suite of signaling systems is ubiquitous in both arteries and veins in at least this elasmobranch species. Their role in hemodynamics and osmoregulation (perfusion of gill and rectal gland) remains to be determined.     

家蚕神经肽基因的筛查及成熟肽的预测

神经肽（neuropeptide）是家蚕Bombyx mori体内重要的调控物质。为了充分理解神经肽对家蚕发育的调控, 扩充家蚕神经肽的数量, 本研究利用BLAST的tblastn程序结合OpenOffice软件的查找程序, 基于其他昆虫和无脊椎动物神经肽的同源性和保守的结构特点, 在家蚕基因、理论蛋白质数据库及NCBI中进行全面而系统的基因筛查; 并利用各种在线工具对所筛查到基因和理论蛋白质的结构和成熟肽进行预测分析。结果共获得allatostatin-A （AST-A）, allatostatin-B （AST-B）, allatostatin-C （AST-C）, allatropin (AT), ecdysis-triggering hormone （ETH）, crustacean cardioactive peptide (CCAP)和FMRFamide等31个神经肽基因家族, 包括37个神经肽基因亚家族, 共计44个神经肽基因; 预测出193个成熟的神经肽, 其中73个根据家族同源性预测在C末端发生酰胺化, 而6个被预测在N末端发生了环化, 9个被预测酪氨酸发生了硫酸化。大部分成熟神经肽都具有明显的家族结构特征, 但proctolin, CCAP及CAPA-PK成熟肽结构上与其他昆虫相比有所扩展。结果提示, 家蚕神经和内分泌细胞产生了几乎在所有昆虫中具有的神经肽前体; 进化过程中大部分成熟神经肽的氨基酸序列在种间产生了差异, 但家族特征性基序高度保守。本研究为神经肽功能研究以及神经肽对家蚕发育尤其是蛹期发育调控的研究奠定了基础。     

Exon-intron structure of the Xist gene in elephant, armadillo, and the ancestor of placental mammals

The Xist gene belongs to the class of long noncoding regulatory RNA genes which play a key role in the process of inactivation of one of the X chromosomes in females of placental mammals. Based on interspecific comparative sequence analysis performed using a set ofbioinformatic programs and approaches, the exon-intron gene structure was first described in two species, elephant and armadillo, belonging to the most primitive placental mammal groups, Afrotheria and Xenarthra. Using multiple sequence alignment of the species representing all main groups of placental mammals (12 species), consensus sequence of the ancestral gene was reconstructed. In the gene structure four evolutionary conserved regions with the identity level of 90% and the sizes of more than 100 bp were identified. Substantial contribution of transposable elements to the gene origin, as well as mosaic evolution of certain elements of the Xist locus was demonstrated. It is likely that the ancestral gene consisted often exons and was formed before the radiation of placental mammals, in the period from 140 to 105 Myr ago.     

Nonrandom divergence of gene expression following gene and genome duplications in the flowering plant Arabidopsis thaliana

Background  

Genome analyses have revealed that gene duplication in plants is rampant. Furthermore, many of the duplicated genes seem to have been created through ancient genome-wide duplication events. Recently, we have shown that gene loss is strikingly different for large- and small-scale duplication events and highly biased towards the functional class to which a gene belongs. Here, we study the expression divergence of genes that were created during large- and small-scale gene duplication events by means of microarray data and investigate both the influence of the origin (mode of duplication) and the function of the duplicated genes on expression divergence.     

World phylogeography and male‐mediated gene flow in the sandbar shark,Carcharhinus plumbeus

The sandbar shark, Carcharhinus plumbeus, is a large, cosmopolitan, coastal species. Females are thought to show philopatry to nursery grounds while males potentially migrate long distances, creating an opportunity for male‐mediated gene flow that may lead to discordance in patterns revealed by mitochondrial DNA (mtDNA) and nuclear markers. While this dynamic has been investigated in elasmobranchs over small spatial scales, it has not been examined at a global level. We examined patterns of historical phylogeography and contemporary gene flow by genotyping 329 individuals from nine locations throughout the species’ range at eight nuclear microsatellite markers and sequencing the complete mtDNA control region. Pairwise comparisons often resulted in fixation indices and divergence estimates of greater magnitude using mtDNA sequence data than microsatellite data. In addition, multiple methods of estimation suggested fewer populations based on microsatellite loci than on mtDNA sequence data. Coalescent analyses suggest divergence and restricted migration among Hawaii, Taiwan, eastern and western Australia using mtDNA sequence data and no divergence and high migration rates, between Taiwan and both Australian sites using microsatellite data. Evidence of secondary contact was detected between several localities and appears to be discreet in time rather than continuous. Collectively, these data suggest complex spatial/temporal relationships between shark populations that may feature pulses of female dispersal and more continuous male‐mediated gene flow.