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In gnathostomes, the adenohypophysis is believed to develop through hierarchically organized epigenetic interactions based primarily on the topographical relationships between tissues. FGF and BMP signaling molecules derived from hypothalamic and adenohypophysial anlage are cooperatively involved in the pituitary organogenesis. From a comparison of developmental processes and gene expression patterns of pituitary-related genes between lamprey (Lethenteron japonicum) and gnathostomes, we speculate on the evolutionary pathway of the vertebrate adenohypophysis. In the lamprey, the adenohypophysis was derived from the nasohypophysial placode (NHP) that developed anterior to the oral ectoderm. The NHP was identified by the expression of LjPitxA, before actual histogenesis, but it was initially distant from the future hypothalamic region. Subsequently, the NHP expressed both LjFgf8/17 and LjBmp2/4a gene transcripts, whereas no signaling molecules could be identified in the hypothalamic anlage. The NHP grew caudally to establish a de novo contact with the hypothalamic region by the midpharyngula stage, and gave rise to both the adenohypophysis and an unpaired nasal organ. Thus, the topographical relationship between the NHP and the hypothalamic region is established secondarily in the lamprey, unlike gnathostomes. Comparing the developmental pattern of the adenohypophysial homologue in amphioxus, we hypothesize that a modification of the regulation of the growth factor encoding gene may have been involved above heterochrony and heterotopy.

Abstract Background The number of members of the Dlx gene family increased during the two rounds of whole-genome duplication that occurred in the common ancestor of the vertebrates. Because the Dlx genes are involved in the development of the cranial skeleton, brain, and sensory organs, their expression patterns have been analysed in various organisms in the context of evolutionary developmental biology. Six Dlx genes have been isolated in the lampreys, a group of living jawless vertebrates (cyclostomes), and their expression patterns analysed. However, little is known about the Dlx genes in the hagfish, the other cyclostome group, mainly because the embryological analysis of this animal is difficult. Results To identify the hagfish Dlx genes and describe their expression patterns, we cloned the cDNA from embryos of the Japanese inshore hagfish Eptatretus burgeri. Our results show that the hagfish has at least six Dlx genes and one pseudogene. In a phylogenetic analysis, the hagfish Dlx genes and those of the lampreys tended to be excluded from the clade of the gnathostome Dlx genes. In several cases, the lamprey Dlx genes clustered with the clade consisting of two hagfish genes, suggesting that independent gene duplications have occurred in the hagfish lineage. Analysis of the expression of these genes showed distinctive overlapping expression patterns in the cranial mesenchymal cells and the inner ear. Conclusions Independent duplication, pseudogenization, and loss of the Dlx genes probably occurred in the hagfish lineage after its split from the other vertebrate lineages. This pattern is reminiscent of the non-parsimonious evolution of its morphological traits, including its inner ear and vertebrae, which indicate that this group is an early-branching lineage that diverged before those characters evolved.

Abstract Background Extant sauropsids (reptiles and birds) are divided into two major lineages, the lineage of Testudines (turtles) and Archosauria (crocodilians and birds) and the lineage of Lepidosauria (tuatara, lizards, worm lizards and snakes). Karyotypes of these sauropsidan groups generally consist of macrochromosomes and microchromosomes. In chicken, microchromosomes exhibit a higher GC-content than macrochromosomes. To examine the pattern of intra-genomic GC heterogeneity in lepidosaurian genomes, we constructed a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 183 cDNA clones by fluorescence in situ hybridization, and examined the correlation between the GC-content of exonic third codon positions (GC3) of the genes and the size of chromosomes on which the genes were localized. Results Although GC3 distribution of snake genes was relatively homogeneous compared with those of the other amniotes, microchromosomal genes showed significantly higher GC3 than macrochromosomal genes as in chicken. Our snake cytogenetic map also identified several conserved segments between the snake macrochromosomes and the chicken microchromosomes. Cross-species comparisons revealed that GC3 of most snake orthologs in such macrochromosomal segments were GC-poor (GC3 < 50%) whereas those of chicken orthologs in microchromosomes were relatively GC-rich (GC3 ≥ 50%). Conclusion Our results suggest that the chromosome size-dependent GC heterogeneity had already occurred before the lepidosaur-archosaur split, 275 million years ago. This character was probably present in the common ancestor of lepidosaurs and but lost in the lineage leading to Anolis during the diversification of lepidosaurs. We also identified several genes whose GC-content might have been influenced by the size of the chromosomes on which they were harbored over the course of sauropsid evolution.

Abstract Background In understanding the evolutionary process of vertebrates, cyclostomes (hagfishes and lamprey) occupy crucial positions. Resolving molecular phylogenetic relationships of cyclostome genes with gnathostomes (jawed vertebrates) genes is indispensable in deciphering both the species tree and gene trees. However, molecular phylogenetic analyses, especially those including lamprey genes, have produced highly discordant results between gene families. To efficiently scrutinize this problem using partial genome assemblies of early vertebrates, we focused on the potassium voltage-gated channel, shaker-related (KCNA) family, whose members are mostly single-exon. Results Seven sea lamprey KCNA genes as well as six elephant shark genes were identified, and their orthologies to bony vertebrate subgroups were assessed. In contrast to robustly supported orthology of the elephant shark genes to gnathostome subgroups, clear orthology of any sea lamprey gene could not be established. Notably, sea lamprey KCNA sequences displayed unique codon usage pattern and amino acid composition, probably associated with exceptionally high GC-content in their coding regions. This lamprey-specific property of coding sequences was also observed generally for genes outside this gene family. Conclusions Our results suggest that secondary modifications of sequence properties unique to the lamprey lineage may be one of the factors preventing robust orthology assessments of lamprey genes, which deserves further genome-wide validation. The lamprey lineage-specific alteration of protein-coding sequence properties needs to be taken into consideration in tackling the key questions about early vertebrate evolution.

Abstract Background Cichlid fishes have undergone rapid, expansive evolutionary radiations that are manifested in the diversification of their trophic morphologies, tooth patterning and coloration. Understanding the molecular mechanisms that underlie the cichlids' unique patterns of evolution requires a thorough examination of genes that pattern the neural crest, from which these diverse phenotypes are derived. Among those genes, the homeobox-containing Dlx gene family is of particular interest since it is involved in the patterning of the brain, jaws and teeth. Results In this study, we characterized the dlx genes of an African cichlid fish, Astatotilapia burtoni, to provide a baseline to later allow cross-species comparison within Cichlidae. We identified seven dlx paralogs (dlx1a, -2a, -4a, -3b, -4b, -5a and -6a), whose orthologies were validated with molecular phylogenetic trees. The intergenic regions of three dlx gene clusters (dlx1a-2a, dlx3b-4b, and dlx5a-6a) were amplified with long PCR. Intensive cross-species comparison revealed a number of conserved non-coding elements (CNEs) that are shared with other percomorph fishes. This analysis highlighted additional lineage-specific gains/losses of CNEs in different teleost fish lineages and a novel CNE that had previously not been identified. Our gene expression analyses revealed overlapping but distinct expression of dlx orthologs in the developing brain and pharyngeal arches. Notably, four of the seven A. burtoni dlx genes, dlx2a, dlx3b, dlx4a and dlx5a, were expressed in the developing pharyngeal teeth. Conclusion This comparative study of the dlx genes of A. burtoni has deepened our knowledge of the diversity of the Dlx gene family, in terms of gene repertoire, expression patterns and non-coding elements. We have identified possible cichlid lineage-specific changes, including losses of a subset of dlx expression domains in the pharyngeal teeth, which will be the targets of future functional studies.

Abstract Background Whole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs. Results As a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated bmp16, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of bmp16 to the Bmp2/4 subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a bmp16 ortholog in tetrapods is due to a secondary loss. In situ hybridization showed embryonic expression of the zebrafish bmp16 in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos. Conclusion Comparisons of expression patterns revealed (1) the redundancy of bmp16 expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2) the loss of craniofacial expression and gain of swim bladder expression of bmp16 after the gene duplication between Bmp2, -4 and -16. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.

Abstract Background Phylogenetic analyses of jawed vertebrates based on mitochondrial sequences often result in confusing inferences which are obviously inconsistent with generally accepted trees. In particular, in a hypothesis by Rasmussen and Arnason based on mitochondrial trees, cartilaginous fishes have a terminal position in a paraphyletic cluster of bony fishes. No previous analysis based on nuclear DNA-coded genes could significantly reject the mitochondrial trees of jawed vertebrates. Results We have cloned and sequenced seven nuclear DNA-coded genes from 13 vertebrate species. These sequences, together with sequences available from databases including 13 jawed vertebrates from eight major groups (cartilaginous fishes, bichir, chondrosteans, gar, bowfin, teleost fishes, lungfishes and tetrapods) and an outgroup (a cyclostome and a lancelet), have been subjected to phylogenetic analyses based on the maximum likelihood method. Conclusion Cartilaginous fishes have been inferred to be basal to other jawed vertebrates, which is consistent with the generally accepted view. The minimum log-likelihood difference between the maximum likelihood tree and trees not supporting the basal position of cartilaginous fishes is 18.3 ± 13.1. The hypothesis by Rasmussen and Arnason has been significantly rejected with the minimum log-likelihood difference of 123 ± 23.3. Our tree has also shown that living holosteans, comprising bowfin and gar, form a monophyletic group which is the sister group to teleost fishes. This is consistent with a formerly prevalent view of vertebrate classification, although inconsistent with both of the current morphology-based and mitochondrial sequence-based trees. Furthermore, the bichir has been shown to be the basal ray-finned fish. Tetrapods and lungfish have formed a monophyletic cluster in the tree inferred from the concatenated alignment, being consistent with the currently prevalent view. It also remains possible that tetrapods are more closely related to ray-finned fishes than to lungfishes.

Protogyny, being capable of changing from female to male during their lifetime, is prevalent in 20 families of teleosts but is believed to have evolved within specific evolutionary lineages. Therefore, shared regulatory factors governing the sex change process are expected to be conserved across protogynous fishes. However, a comprehensive understanding of this mechanism remains elusive. To identify these factors, we conducted a meta-analysis using gonadal transcriptome data from seven species. We curated data pairs of ovarian tissue and transitional gonad, and employed ratios of expression level as a unified criterion for differential expression, enabling a meta-analysis across species. Our approach revealed that classical sex change-related genes exhibited differential expression levels between the ovary and transitional gonads, consistent with previous reports. These results validate our methodology's robustness. Additionally, we identified novel genes not previously linked to gonadal sex change in fish. Notably, changes in the expression levels of acetoacetyl-CoA synthetase and apolipoprotein Eb, which are involved in cholesterol synthesis and transport, respectively, suggest that the levels of cholesterol, a precursor of steroid hormones crucial for sex change, are decreased upon sex change onset in the gonads. This implies a potential universal influence of cholesterol dynamics on gonadal transformation in protogyny., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c +/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c +/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10 -16 , binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action., (© 2024. The Author(s).)

Chondrichthyes is an important lineage to reconstruct the evolutionary history of vertebrates. Here, we analyzed genome synteny for six chondrichthyan chromosome-level genomes. Our comparative analysis reveals a slow evolutionary rate of chromosomal changes, with infrequent but independent fusions observed in sharks, skates, and chimaeras. The chondrichthyan common ancestor had a proto-vertebrate-like karyotype, including the presence of 18 microchromosome pairs. The X chromosome is a conversed microchromosome shared by all sharks, suggesting a likely common origin of the sex chromosome at least 181 million years ago. We characterized the Y chromosomes of two sharks that are highly differentiated from the X except for a small young evolutionary stratum and a small pseudoautosomal region. We found that shark sex chromosomes lack global dosage compensation but that dosage-sensitive genes are locally compensated. Our study on shark chromosome evolution enhances our understanding of shark sex chromosomes and vertebrate chromosome evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Improvements in DNA sequencing technology are allowing the dramatic increase of whole genome data for a wide variety of species. Such genome sequence data can assist the monitoring of intraspecific genetic diversity, but is often lacking for threatened species. In this project, we focused on the national Red List, a catalog of extinct and threatened species, issued by the Japanese government. We combined the data included in it with the record of genome assembly in NCBI and tabulated the assembly availability of the species in the list. The combined data shows a low percentage (2.1%) of the availability of whole genome sequence data for the taxa ranked on the Japanese Red List as well as a strong bias towards mammals and birds in Animalia and vascular plants in Plantae. Our data presentation highlights potential systematic limitations in genome sequencing (e.g., budget for sequencing large genomes of amphibians) and instructs future policies including which taxon needs more effort for genome sequencing. The resultant tables are available in the original website https://treethinkers.nig.ac.jp/redlist/ and are regularly updated., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Kryukov K et al.)

In vertebrates, folliculogenesis and ovulation are regulated by two distinct pituitary gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Currently, there is an intriguing consensus that a single hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), regulates the secretion of both FSH and LH, although the required timing and functions of FSH and LH are different. However, recent studies in many non-mammalian vertebrates indicated that GnRH is dispensable for FSH function. Here, by using medaka as a model teleost, we successfully identify cholecystokinin as the other gonadotropin regulator, FSH-releasing hormone (FSH-RH). Our histological and in vitro analyses demonstrate that hypothalamic cholecystokinin-expressing neurons directly affect FSH cells through the cholecystokinin receptor, Cck2rb, thereby increasing the expression and release of FSH. Remarkably, the knockout of this pathway minimizes FSH expression and results in a failure of folliculogenesis. Here, we propose the existence of the "dual GnRH model" in vertebrates that utilize both FSH-RH and LH-RH., (© 2024. The Author(s).)

Homology in vertebrate body plans is traditionally ascribed to the high-level conservation of regulatory components within the genetic programs governing them, particularly during the "phylotypic stage." However, advancements in embryology and molecular phylogeny have unveiled the dynamic nature of gene repertoires responsible for early development. Notably, the Nodal and Lefty genes, members of the transforming growth factor-beta superfamily producing intercellular signaling molecules and crucial for left-right (L-R) symmetry breaking, exhibit distinctive features within their gene repertoires. These features encompass among-species gene repertoire variations resulting from gene gain and loss, as well as gene conversion. Despite their significance, these features have been largely unexplored in a phylogenetic context, but accumulating genome-wide sequence information is allowing the scrutiny of these features. It has exposed hidden paralogy between Nodal1 and Nodal2 genes resulting from differential gene loss in amniotes. In parallel, the tandem cluster of Lefty1 and Lefty2 genes, which was thought to be confined to mammals, is observed in sharks and rays, with an unexpected phylogenetic pattern. This article provides a comprehensive review of the current understanding of the origins of these vertebrate gene repertoires and proposes a revised nomenclature based on the elucidated history of vertebrate genome evolution., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Pacific saury (Cololabis saira) is a commercially important small pelagic fish species in Asia. In this study, we conducted the first-ever whole genome sequencing of this species, with single molecule, real-time (SMRT) sequencing technology. The obtained high-fidelity (HiFi) long-read sequence data, which amount to ~30-folds of its haploid genome size that was measured with quantitative PCR (1.17 Gb), were assembled into contigs. Scaffolding with Hi-C reads yielded a whole genome assembly containing 24 chromosome-scale sequences, with a scaffold N50 length of 47.7 Mb. Screening of repetitive elements including telomeric repeats was performed to characterize possible factors that need to be resolved towards 'telomere-to-telomere' sequencing. The larger genome size than in medaka, a close relative in Beloniformes, is at least partly explained by larger repetitive element quantity, which is reflected in more abundant tRNAs, in the Pacific saury genome. Protein-coding regions were predicted using transcriptome data, which resulted in 22,274 components. Retrieval of Pacific saury homologs of aquaporin (AQP) genes known from other teleost fishes validated high completeness and continuity of the genome assembly. These resources are available at https://treethinkers.nig.ac.jp/saira/ and will assist various molecular-level studies in fishery science and comparative biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Taste is a vital chemical sense for feeding behaviour. In mammals, the umami and sweet taste receptors comprise three members of the taste receptor type 1 (T1R/TAS1R) family: T1R1, T1R2 and T1R3. Because their functional homologues exist in teleosts, only three TAS1R genes generated by gene duplication are believed to have been inherited from the common ancestor of bony vertebrates. Here, we report five previously uncharacterized TAS1R members in vertebrates, TAS1R4, TAS1R5, TAS1R6, TAS1R7 and TAS1R8, based on genome-wide survey of diverse taxa. We show that mammalian and teleost fish TAS1R2 and TAS1R3 genes are paralogues. Our phylogenetic analysis suggests that the bony vertebrate ancestor had nine TAS1Rs resulting from multiple gene duplications. Some TAS1Rs were lost independently in descendent lineages resulting in retention of only three TAS1Rs in mammals and teleosts. Combining functional assays and expression analysis of non-teleost fishes we show that the novel T1Rs form heterodimers in taste-receptor cells and recognize a broad range of ligands such as essential amino acids, including branched-chain amino acids, which have not been previously considered as T1R ligands. This study reveals diversity of taste sensations in both modern vertebrates and their ancestors, which might have enabled vertebrates to adapt to diverse habitats on Earth., (© 2023. The Author(s).)

The diversity of neural stem cells is a hallmark of the cerebral cortex development in gyrencephalic mammals, such as Primates and Carnivora. Among them, ferrets are a good model for mechanistic studies. However, information on their neural progenitor cells (NPC), termed radial glia (RG), is limited. Here, we surveyed the temporal series of single-cell transcriptomes of progenitors regarding ferret corticogenesis and found a conserved diversity and temporal trajectory between human and ferret NPC, despite the large timescale difference. We found truncated RG (tRG) in ferret cortical development, a progenitor subtype previously described in humans. The combination of in silico and in vivo analyses identified that tRG differentiate into both ependymal and astrogenic cells. Via transcriptomic comparison, we predict that this is also the case in humans. Our findings suggest that tRG plays a role in the formation of adult ventricles, thereby providing the architectural bases for brain expansion., Competing Interests: MB, QW, TS, AS, YT, TS, MK, ON, HK, FM No competing interests declared, SK Reviewing editor, eLife, (© 2023, Bilgic et al.)

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum , an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality., (© 2023 Yamaguchi et al.; Published by Cold Spring Harbor Laboratory Press.)

Gse1 is a component of the CoREST complex that acts as an H3K4 and H3K9 demethylase and regulates gene expression. Here, we examined the expression and role of Gse1 in mouse development. Gse1 is expressed in male and female germ cells and plays both maternal and zygotic roles. Thus, maternal deletion of Gse1 results in a high incidence of prenatal death, and zygotic deletion leads to embryonic lethality from embryonic day 12.5 (E12.5) and perinatal death. Gse1 is expressed in the junctional zone and the labyrinth of the developing placenta. Gse1 mutant (Gse1 Δex3/Δex3 ) placenta begins to exhibit histological defects from E14.5, being deficient in MCT4 + syncytiotrophoblast II. The number of various cell types was largely maintained in the mutant placenta at E10.5, but several genes were upregulated in giant trophoblasts at E10.5. Placenta-specific deletion of Gse1 with Tat-Cre suggested that defects in Gse1 Δex3/Δex3 embryos are due to placental function deficiency. These results suggest that Gse1 is required for placental development in mice, and in turn, is essential for embryonic development., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The presentation of nicotinic acetylcholine receptors (nAChRs) on synaptic membranes is crucial for generating cholinergic circuits, some of which are associated with memory function and neurodegenerative disorders. Although the physiology and structure of nAChR, a cation channel comprising five subunits, have been extensively studied, little is known about how the receptor levels in interneuronal synapses are determined and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices and intracellular proteins. By a genetic screen of Drosophila , we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig , which encodes a secretory matrix protein localized to cholinergic synaptic clefts in the brain. Only the loss of function of Dα5 among the 10 nAChR subunits suppressed hig mutant phenotypes in both male and female flies. Dα5 behaved as a lethal factor when Hig was defective; loss of Dα5 in hig mutants rescued lethality, upregulating Dα6 synaptic levels. By contrast, levels of Dα5, Dα6, and Dα7 subunits were all reduced in hig mutants. These three subunits have distinct properties for interaction with Hig or trafficking, as confirmed by chimeric subunit experiments. Notably, the chimeric Dα5 protein, which has the extracellular sequences that display no positive interaction with Hig, exhibited abnormal distribution and lethality even in the presence of Hig. We propose that the sequestering subunit Dα5 functions by reducing synaptic levels of nAChR through internalization, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix. SIGNIFICANCE STATEMENT Because the cholinergic synapse is one of the major synapses that generate various brain functions, numerous studies have sought to reveal the physiology and structure of the nicotinic acetylcholine receptor (nAChR). However, little is known about how synaptic levels of nAChR are controlled and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices. By a genetic screen of Drosophila , we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig , which encodes a secretory matrix protein localized to cholinergic synaptic clefts. Our data indicate that Dα5 functions in reducing synaptic levels of nAChR, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix., (Copyright © 2023 the authors.)

Functionally indispensable genes are likely to be retained and otherwise to be lost during evolution. This evolutionary fate of a gene can also be affected by factors independent of gene dispensability, including the mutability of genomic positions, but such features have not been examined well. To uncover the genomic features associated with gene loss, we investigated the characteristics of genomic regions where genes have been independently lost in multiple lineages. With a comprehensive scan of gene phylogenies of vertebrates with a careful inspection of evolutionary gene losses, we identified 813 human genes whose orthologs were lost in multiple mammalian lineages: designated 'elusive genes.' These elusive genes were located in genomic regions with rapid nucleotide substitution, high GC content, and high gene density. A comparison of the orthologous regions of such elusive genes across vertebrates revealed that these features had been established before the radiation of the extant vertebrates approximately 500 million years ago. The association of human elusive genes with transcriptomic and epigenomic characteristics illuminated that the genomic regions containing such genes were subject to repressive transcriptional regulation. Thus, the heterogeneous genomic features driving gene fates toward loss have been in place and may sometimes have relaxed the functional indispensability of such genes. This study sheds light on the complex interplay between gene function and local genomic properties in shaping gene evolution that has persisted since the vertebrate ancestor., Competing Interests: YH No competing interests declared, SK Reviewing editor, eLife, (© 2023, Hara and Kuraku.)

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca 2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with secondarily expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter feeding, was previously shown to possess the 'blue-shifted' rhodopsin (RHO), which is a signature of deep-sea adaptation. In this study, our spectroscopy of recombinant whale shark RHO mutants revealed that this blue shift is caused dominantly by an unprecedented spectral tuning site 94. In humans, the mutation at the site causes congenital stationary night blindness (CSNB) by reducing the thermal stability of RHO. Similarly, the RHO of deep-diving whale shark has reduced thermal stability, which was experimentally shown to be achieved by site 178 and 94. RHOs having the natural substitution at site 94 are also found in some Antarctic fishes, suggesting that the blue shift by the substitution at the CSNB site associated with the reduction in thermal stability might be allowed in cold-water deep-sea habitats.

Reproductive modes of vertebrates are classified into two major embryonic nutritional types: yolk deposits (i.e., lecithotrophy) and maternal investment (i.e., matrotrophy). Vitellogenin (VTG), a major egg yolk protein synthesized in the female liver, is one of the molecules relevant to the lecithotrophy-to-matrotrophy shift in bony vertebrates. In mammals, all VTG genes are lost following the lecithotrophy-to-matrotrophy shift, and it remains to be elucidated whether the lecithotrophy-to-matrotrophy shift in nonmammalians is also associated with VTG repertoire modification. In this study, we focused on chondrichthyans (cartilaginous fishes)-a vertebrate clade that underwent multiple lecithotrophy-to-matrotrophy shifts. For an exhaustive search of homologs, we performed tissue-by-tissue transcriptome sequencing for two viviparous chondrichthyans, the frilled shark Chlamydoselachus anguineus and the spotless smooth-hound Mustelus griseus, and inferred the molecular phylogeny of VTG and its receptor very low-density lipoprotein receptor (VLDLR), across diverse vertebrates. As a result, we identified either three or four VTG orthologs in chondrichthyans including viviparous species. We also showed that chondrichthyans had two additional VLDLR orthologs previously unrecognized in their unique lineage (designated as VLDLRc2 and VLDLRc3). Notably, VTG gene expression patterns differed in the species studied depending on their reproductive mode; VTGs are broadly expressed in multiple tissues, including the uterus, in the two viviparous sharks, and in addition to the liver. This finding suggests that the chondrichthyans VTGs do not only function as the yolk nutrient but also as the matrotrophic factor. Altogether, our study indicates that the lecithotrophy-to-matrotrophy shift in chondrichthyans was achieved through a distinct evolutionary process from mammals., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles., (© 2023 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

Homeostatic control and reproductive functions of humans are regulated at the molecular levels largely by peptide hormones secreted from endocrine and/or neuroendocrine cells in the central nervous system and peripheral organs. Homologs of those hormones and their receptors function similarly in many vertebrate species distantly related to humans, but the evolutionary history of the endocrine system involving those factors has been obscured by the scarcity of genome DNA sequence information of some taxa that potentially contain their orthologs. Focusing on non-osteichthyan vertebrates, namely jawless and cartilaginous fishes, this article illustrates how investigating genome sequence information assists our understanding of the diversification of vertebrate gene repertoires in four broad themes: ( a ) the presence or absence of genes, ( b ) multiplication and maintenance of paralogs, ( c ) differential fates of duplicated paralogs, and ( d ) the evolutionary timing of gene origins.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Nishimura O et al.)

Most cartilaginous fishes live in seawater (SW), but a few exceptional elasmobranchs (sharks and rays) are euryhaline and can acclimate to freshwater (FW) environments. The plasma of elasmobranchs is high in NaCl and urea concentrations, which constrains osmotic water loss. However, these euryhaline elasmobranchs maintain high levels of plasma NaCl and urea even when acclimating to low salinity, resulting in a strong osmotic gradient from external environment to body fluid. The kidney consequently produces a large volume of dilute urine to cope with the water influx. In the present study, we investigated the molecular mechanisms of dilute urine production in the kidney of Japanese red stingray, Hemitrygon akajei , transferred from SW to low-salinity environments. We showed that red stingray maintained high plasma NaCl and urea levels by reabsorbing more osmolytes in the kidney when transferred to low salinity. RNA-seq and qPCR analyses were conducted to identify genes involved in NaCl and urea reabsorption under the low-salinity conditions, and the upregulated gene expressions of Na + -K + -Cl - cotransporter 2 ( nkcc2 ) and Na + /K + -ATPase ( nka ) were found in the FW-acclimated individuals. These upregulations occurred in the early distal tubule (EDT) in the bundle zone of the kidney, which coils around the proximal and collecting tubules to form the highly convoluted structure of batoid nephron. Considering the previously proposed model for urea reabsorption, the upregulation of nkcc2 and nka not only causes the reabsorption of NaCl in the EDT, but potentially also supports enhanced urea reabsorption and eventually the production of dilute urine in FW-acclimated individuals. We propose advantageous characteristics of the batoid-type nephron that facilitate acclimation to a wide range of salinities, which might have allowed the batoids to expand their habitats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aburatani, Takagi, Wong, Kuraku, Tanegashima, Kadota, Saito, Godo, Sakamoto and Hyodo.)

Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex-determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene spans ∼100 kb and is flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates.

The vomeronasal type 2 receptor (V2R, also called OlfC) multigene family is found in a broad range of jawed vertebrates from cartilaginous fish to tetrapods. V2Rs encode receptors for food-related amino acids in teleost fish, whereas for peptide pheromones in mammals. In addition, V2Rs of teleost fish are phylogenetically distinct from those of tetrapods, implying a drastic change in the V2R repertoire during terrestrial adaptation. To understand the process of diversification of V2Rs in vertebrates from "fish-type" to "tetrapod-type", we conducted an exhaustive search for V2Rs in cartilaginous fish (chimeras, sharks, and skates) and basal ray-finned fish (reedfish, sterlet, and spotted gar), and compared them with those of teleost, coelacanth, and tetrapods. Phylogenetic and synteny analyses on 1897 V2Rs revealed that basal ray-finned fish possess unexpectedly higher number of V2Rs compared with cartilaginous fish, implying that V2R gene repertoires expanded in the common ancestor of Osteichthyes. Furthermore, reedfish and sterlet possessed various V2Rs that belonged to both "fish-type" and "tetrapod-type", suggesting that the common ancestor of Osteichthyes possess "tetrapod-type" V2Rs although they inhabited underwater environments. Thus, the unexpected diversity of V2Rs in basal ray-finned fish may provide insight into how the olfaction of osteichthyan ancestors adapt from water to land., (© 2022. The Author(s).)

Forms of embryonic nutrition are highly diverse in cartilaginous fishes, which contain oviparity, yolk-sac viviparity and several types of matrotrophic viviparity (histotrophy, oophagy, and placentotrophy). The molecular mechanisms of embryonic nutrition are poorly understood in these animals as few species are capable of reproducing in captivity. Oviparous cartilaginous fishes solely depend on yolk nutrients for their growth and development. In the present study, we compared the contribution to embryonic nutrition of the embryonic intestine with the yolk sac membrane (YSM). RNA-seq analysis was performed on the embryonic intestine and YSM of the oviparous cloudy catshark Scyliorhinus torazame to identify candidate genes involved in nutrient metabolism to further the understanding of nutrient utilization of developing embryos. RNA-seq discovery was subsequently confirmed by quantitative PCR analysis and we identified increases in several amino acid transporter genes (slc3a1, slc6a19, slc3a2, slc7a7) as well as genes involved in lipid absorption (apob and mtp) in the intestine after 'pre-hatching', which is a developmental event marked by an early opening of the egg case about 4 months before hatching. Although a reciprocal decrease in the nutritional role of YSM was expected after the intestine became functional, we observed similar increases in gene expression among amino acid transporters, lipid absorption molecules, and lysosomal cathepsins in the extraembryonic YSM in late developmental stages. Ultrastructure of the endodermal cells of YSM showed that yolk granules were incorporated by endocytosis, and the number of granules increased during development. Furthermore, the digestion of yolk granules in the YSM and nutrient transport through the basolateral membrane of the endodermal cells appeared to be enhanced after pre-hatching. These findings suggest that nutrient digestion and absorption is highly activated in both intestine and YSM after pre-hatching in catshark embryos, which supports the rapid growth at late developmental stages., Competing Interests: The authors have declared that no competing interests exist.

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Competing Interests: The authors declare no competing interest.

The notochord functions primarily as a supporting tissue to maintain the anteroposterior axis of primitive chordates, a function that is replaced entirely by the vertebral column in many vertebrates. The notochord still appears during vertebrate embryogenesis and plays a crucial role in the developmental pattern formation of surrounding structures, such as the somites and neural tube, providing the basis for the vertebrate body plan. The indispensable role of the notochord has often been referred to as the developmental burden and used to explain the evolutionary conservation of notochord; however, the existence of this burden has not been successfully exemplified so far. Since the adaptive value of target tissues appears to result in the evolutionary conservation of upstream structures through the developmental burden, we performed comparative gene expression profiling of the notochord, somites, and neural tube during the mid-embryonic stages in turtles and chicken to measure their evolutionary conservation. When compared with the somites and neural tube, overall gene expression profiles in the notochord showed significantly lower or merely comparable levels of conservation. However, genes involved in inductive signalings, such as the sonic hedgehog (Shh) cascade and the formation of functional primary cilia, showed relatively higher levels of conservation in all the three structures analyzed. Collectively, these results suggest that shh signals are critical as the inductive source and receiving structures, possibly constituting the inter-dependencies of developmental burden., (© 2021 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

Three types of variable lymphocyte receptor (VLR) genes, VLRA , VLRB , and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell-like, while VLRB-expressing cells are B cell-like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey., Competing Interests: Competing interest statement: M.D.C. is a cofounder and shareholder of NovAb, Inc., which produces lamprey antibodies for biomedical purposes, and J.P.R. is a consultant for NovAb. One of the reviewers (T.B.) and three of the authors (S.D., M.H., and M.D.C.) contributed to a review article published online in 2017 (14).

The recent development of ecological studies has been fueled by the introduction of massive information based on chromosome-scale genome sequences, even for species for which genetic linkage is not accessible. This was enabled mainly by the application of Hi-C, a method for genome-wide chromosome conformation capture that was originally developed for investigating the long-range interaction of chromatins. Performing genomic scaffolding using Hi-C data is highly resource-demanding and employs elaborate laboratory steps for sample preparation. It starts with building a primary genome sequence assembly as an input, which is followed by computation for genome scaffolding using Hi-C data, requiring careful validation. This article presents technical considerations for obtaining optimal Hi-C scaffolding results and provides a test case of its application to a reptile species, the Madagascar ground gecko (Paroedura picta). Among the metrics that are frequently used for evaluating scaffolding results, we investigate the validity of the completeness assessment of chromosome-scale genome assemblies using single-copy reference orthologues., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Globin-X (GbX) is an enigmatic member of the vertebrate globin gene family with a wide phyletic distribution that spans protostomes and deuterostomes. Unlike canonical globins such as hemoglobins and myoglobins, functional data suggest that GbX does not have a primary respiratory function. Instead, evidence suggests that the monomeric, membrane-bound GbX may play a role in cellular signaling or protection against the oxidation of membrane lipids. Recently released genomes from key vertebrates provide an excellent opportunity to address questions about the early stages of the evolution of GbX in vertebrates. We integrate bioinformatics, synteny, and phylogenetic analyses to characterize the diversity of GbX genes in nonteleost ray-finned fishes, resolve relationships between the GbX genes of cartilaginous fish and bony vertebrates, and demonstrate that the GbX genes of cyclostomes and gnathostomes derive from independent duplications. Our study highlights the role that whole-genome duplications (WGDs) have played in expanding the repertoire of genes in vertebrate genomes. Our results indicate that GbX paralogs have a remarkably high rate of retention following WGDs relative to other globin genes and provide an evolutionary framework for interpreting results of experiments that examine functional properties of GbX and patterns of tissue-specific expression. By identifying GbX paralogs that are products of different WGDs, our results can guide the design of experimental work to explore whether gene duplicates that originate via WGDs have evolved novel functional properties or expression profiles relative to singleton or tandemly duplicated copies of GbX., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Developmental studies of sharks and rays (elasmobranchs) have provided much insight into the process of morphological evolution of vertebrates. Although those studies are supposedly fueled by large-scale molecular sequencing information, whole-genome sequences of sharks and rays were made available only recently. One compelling difficulty of elasmobranch developmental biology is the low accessibility to embryonic study materials and their slow development. Another limiting factor is the relatively large size of their genomes. Moreover, their large body sizes restrict sustainable captive breeding, while their high body fluid osmolarity prevents reproducible cell culturing for in vitro experimentation, which has also limited our knowledge of their chromosomal organization for validation of genome sequencing products. This article focuses on egg-laying elasmobranch species used in developmental biology and provides an overview of the characteristics of the shark and ray genomes revealed to date. Developmental studies performed on a gene-by-gene basis are also reviewed from a whole-genome perspective. Among the popular regulatory genes studied in developmental biology, I scrutinize shark homologs of Wnt genes that highlight vanishing repertoires in many other vertebrate lineages, as well as Hox genes that underwent an unexpected modification unique to the elasmobranch lineage. These topics are discussed together with insights into the reconstruction of developmental programs in the common ancestor of vertebrates and its subsequent evolutionary trajectories that mark the features that are unique to, and those characterizing the diversity among, cartilaginous fishes., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Chondrichthyes (cartilaginous fishes) are fundamental for understanding vertebrate evolution, yet their genomes are understudied. We report long-read sequencing of the whale shark genome to generate the best gapless chondrichthyan genome assembly yet with higher contig contiguity than all other cartilaginous fish genomes, and studied vertebrate genomic evolution of ancestral gene families, immunity, and gigantism. We found a major increase in gene families at the origin of gnathostomes (jawed vertebrates) independent of their genome duplication. We studied vertebrate pathogen recognition receptors (PRRs), which are key in initiating innate immune defense, and found diverse patterns of gene family evolution, demonstrating that adaptive immunity in gnathostomes did not fully displace germline-encoded PRR innovation. We also discovered a new toll-like receptor (TLR29) and three NOD1 copies in the whale shark. We found chondrichthyan and giant vertebrate genomes had decreased substitution rates compared to other vertebrates, but gene family expansion rates varied among vertebrate giants, suggesting substitution and expansion rates of gene families are decoupled in vertebrate genomes. Finally, we found gene families that shifted in expansion rate in vertebrate giants were enriched for human cancer-related genes, consistent with gigantism requiring adaptations to suppress cancer., Competing Interests: MT, AR, HD, RN, KS, SK, AP, AD, TR No competing interests declared, SK Reviewing editor, eLife

The vertebrate cerebellum arises at the dorsal part of rhombomere 1, induced by signals from the isthmic organizer. Two major cerebellar neuronal subtypes, granule cells (excitatory) and Purkinje cells (inhibitory), are generated from the anterior rhombic lip and the ventricular zone, respectively. This regionalization and the way it develops are shared in all extant jawed vertebrates (gnathostomes). However, very little is known about early evolution of the cerebellum. The lamprey, an extant jawless vertebrate lineage or cyclostome, possesses an undifferentiated, plate-like cerebellum, whereas the hagfish, another cyclostome lineage, is thought to lack a cerebellum proper. In this study, we found that hagfish Atoh1 and Wnt1 genes are co-expressed in the rhombic lip, and Ptf1a is expressed ventrally to them, confirming the existence of r1's rhombic lip and the ventricular zone in cyclostomes. In later stages, lamprey Atoh1 is downregulated in the posterior r1, in which the NeuroD increases, similar to the differentiation process of cerebellar granule cells in gnathostomes. Also, a continuous Atoh1 -positive domain in the rostral r1 is reminiscent of the primordium of valvula cerebelli of ray-finned fishes. Lastly, we detected a GAD -positive domain adjacent to the Ptf1a -positive ventricular zone in lampreys, suggesting that the Ptf1a -positive cells differentiate into some GABAergic inhibitory neurons such as Purkinje and other inhibitory neurons like in gnathostomes. Altogether, we conclude that the ancestral genetic programs for the formation of a distinct cerebellum were established in the last common ancestor of vertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sugahara, Pascual-Anaya, Kuraku, Kuratani and Murakami.)