Yasuhiro Go

Learned behavior, a fundamental adaptive trait in fluctuating environments, is shaped by species-specific constraints. This phenomenon is evident in songbirds, which acquire their species-specific songs through vocal learning. To explore the neurogenetic mechanisms underlying species-specific song learning, we generated F1 hybrid songbirds by crossing Taeniopygia guttata with Aidemosyne modesta. These F1 hybrids demonstrate expanded learning capacities, adeptly mimicking songs from both parental species and other heterospecific songs more extensively than their parental counterparts. Despite the conserved size of brain regions and neuron numbers in the neural circuits for song learning and production, single-cell transcriptomics reveals distinctive transcriptional characteristics in the F1 hybrids, especially in vocal-motor projection neurons. These neurons exhibit enrichment for nonadditively expressed genes, particularly those related to ion channel activity and cell adhesion, which are associated with the degree of song learning among F1 individuals. Our findings provide insights into the emergence of altered learning capabilities through hybridization, linked to cell type-specific transcriptional changes.

Hippocampal pyramidal neurons exhibit diverse spike patterns and gene expression profiles. However, their relationships with single neurons are not fully understood. In this study, we designed an electrophysiology-based experimental procedure to identify gene expression profiles using RNA sequencing of single hippocampal pyramidal neurons whose spike patterns were recorded in living mice. This technique involves a sequence of experiments consisting of in vivo juxtacellular recording and labeling, brain slicing, cell collection, and transcriptome analysis. We demonstrated that the expression levels of a subset of genes in individual hippocampal pyramidal neurons were significantly correlated with their spike burstiness, submillisecond-level spike rise times or spike rates, directly measured by in vivo electrophysiological recordings. Because this methodological approach can be applied across a wide range of brain regions, it is expected to contribute to studies on various neuronal heterogeneities to understand how physiological spike patterns are associated with gene expression profiles.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

The development of individuality during learned behavior is a common trait observed across animal species; however, the underlying biological mechanisms remain understood. Similar to human speech, songbirds develop individually unique songs with species-specific traits through vocal learning. In this study, we investigate the developmental and molecular mechanisms underlying individuality in vocal learning by utilizing F ₁ hybrid songbirds ( Taeniopygia guttata cross with Taeniopygia bichenovii ), taking an integrating approach combining experimentally controlled systematic song tutoring, unbiased discriminant analysis of song features, and single-cell transcriptomics. When tutoring with songs from both parental species, F ₁ hybrid individuals exhibit evident diversity in their acquired songs. Approximately 30% of F ₁ hybrids selectively learn either song of the two parental species, while others develop merged songs that combine traits from both species. Vocal acoustic biases during vocal babbling initially appear as individual differences in songs among F ₁ juveniles and are maintained through the sensitive period of song vocal learning. These vocal acoustic biases emerge independently of the initial auditory experience of hearing the biological father’s and passive tutored songs. We identify individual differences in transcriptional signatures in a subset of cell types, including the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei, which are associated with variations of vocal acoustic features. These findings suggest that a genetically predisposed vocal motor bias serves as the initial origin of individual variation in vocal learning, influencing learning constraints and preferences.

Abstract Psychophysiological disorders chronically impair brain functions, often accompanied by dysregulation of multiple genes, suggesting a multifaceted etiology behind the symptoms. To explore transcription factors (TFs) involved in such transcriptomic changes, we analyzed TF-activity profiles (TFAPs) from the brains of mice experienced chronic stress, and revealed alteration in TF-activity correlating with their pathophysiological phenotypes. We identified REST/NRSF and TCF/LEF associated with depressive phenotypes and discovered that neuropsychiatric drugs sertraline and lithium influence REST- and TCF/LEF-activity, both in vitro and in vivo, thereby affecting gene expression profiles. Pharmacological or genetic manipulation of REST- or TCF/LEF-activity in defeated mice impacts post-stress recovery from depressive phenotypes, with combined treatment further augmenting the outcomes. Our TFAP analysis enhances understanding of molecular mechanisms underpinning chronic diseases, aiding future therapeutic strategy development.

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits.

Because speciation might have been promoted by ancient introgression from an extinct lineage, it is important to detect the existence of 'ghost introgression' in focal taxa and examine its contribution to their diversification. In this study, we examined possible ghost introgression and its contributions to the diversification of ricefishes of the genus Adrianichthys in Lake Poso, an ancient lake on Sulawesi Island, in which some extinctions are known to have occurred. Population-genomic analysis revealed that two extant Adrianichthys species, A. oophorus and A. poptae are reproductively isolated from each other. Comparisons of demographic models demonstrated that introgression from a ghost population, which diverged from the common ancestor of A. oophorus and A. poptae, is essential for reconstructing the demographic history of Adrianichthys. The best model estimated that the divergence of the ghost population greatly predated the divergence between A. oophorus and A. poptae, and that the ghost population secondarily contacted the two extant species within Lake Poso more recently. Genome scans and simulations detected a greatly divergent locus, which cannot be explained without ghost introgression. This locus was also completely segregated between A. oophorus and A. poptae. These findings suggest that variants that came from a ghost population have contributed to the divergence between A. oophorus and A. poptae, but the large time-lag between their divergence and ghost introgression indicates that the contribution of introgression may be restricted.

<jats:title>Abstract</jats:title><jats:p>Given their high sociality and close evolutionary distance to humans, monkeys are an essential animal model for unraveling the biological mechanisms underlying human social behavior and elucidating the pathogenesis of diseases exhibiting abnormal social behavior. However, behavioral analysis of naturally behaving monkeys requires manual counting of various behaviors, which has been a bottleneck due to problems in throughput and objectivity. Here, we developed a three-dimensional markerless motion capture system that utilized multi-view data for robust tracking of individual monkeys and accurate reconstruction of the three-dimensional poses of multiple monkeys living in groups. Validation analysis in two monkey groups revealed that the system enabled the characterization of individual social dispositions and relationships through automated detection of various social events. Analyses of social looking facilitated the investigation of adaptive behaviors in a social group. These results suggest that this motion capture system will significantly enhance our ability to analyze primate social behavior.</jats:p>

<jats:title>Abstract</jats:title> <jats:p>The emergence of individuality during learned behavior is a general feature of animal species, yet the biological bases of its development remain unknown. Similar to human speech, songbirds develop individually-unique songs with species-specific traits through vocal learning. By taking advantage of songbirds as a model system for studying the neural basis of vocal learning and development, we utilized F<jats:sub>1</jats:sub> hybrid songbirds (<jats:italic>Taeniopygia guttata</jats:italic> cross with <jats:italic>T. bichenovii</jats:italic>) to examine the developmental and molecular mechanisms underlying individuality in vocal learning. When tutoring with songs from both parental species, F<jats:sub>1</jats:sub> pupils showed vast individual differences in their acquired songs. Approximately 30% of F<jats:sub>1</jats:sub> hybrids selectively learned either song of the two parental species, whereas others developed merged songs between the parental species. Vocal acoustic biases during vocal babbling were initially observed as individual differences in songs among F<jats:sub>1</jats:sub> juveniles, which were maintained through the sensitive period of song vocal learning. These individual differences in vocal acoustic biases appeared independently from the auditory experience of hearing biological farther’s and passive tutored songs. Furthermore, the idiosyncratic traits of F<jats:sub>1</jats:sub> hybrids’ songs were not correlated with peripheral vocal organ morphology. However, we identified unique transcriptional signatures from the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei associated with individual differences in the acoustic vocal biases, even at the initial stage of vocal learning. These results indicate that a predisposed motor bias influences the individuality observed when learning new motor skills.</jats:p>

PURPOSE: Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. METHODS: Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines. RESULTS: In all four cases, major immune cells, such as T cells (15.1-47.7%), B cells (3.1-19.0%), monocytes (16.5-31.2%), macrophages (1.6-17.4%), dendritic cells (2.4-12.2%), and natural killer cells (7.5-12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells. CONCLUSION: Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

Both Achilles and masticatory muscle tendons are large load-bearing structures, and excessive mechanical loading leads to hypertrophic changes in these tendons. In the maxillofacial region, hyperplasia of the masticatory muscle tendons and aponeurosis affect muscle extensibility resulting in limited mouth opening. Although gene expression profiles of Achilles and patellar tendons under mechanical strain are well investigated in rodents, the gene expression profile of the masticatory muscle tendons remains unexplored. Herein, we examined the gene expression pattern of masticatory muscle tendons and compared it with that of Achilles tendons under tensile strain conditions in the Japanese macaque Macaca fuscata. Primary tenocytes isolated from the masticatory muscle tendons (temporal tendon and masseter aponeurosis) and Achilles tendons were mechanically loaded using the tensile force and gene expression was analyzed using the next-generation sequencing. In tendons exposed to tensile strain, we identified 1076 differentially expressed genes with a false discovery rate (FDR) < 10-10. To identify genes that are differentially expressed in temporal tendon and masseter aponeurosis, an FDR of < 10-10 was used, whereas the FDR for Achilles tendons was set at > 0.05. Results showed that 147 genes are differentially expressed between temporal tendons and masseter aponeurosis, out of which, 125 human orthologs were identified using the Ensemble database. Eight of these orthologs were related to tendons and among them the expression of the glycoprotein nmb and sphingosine kinase 1 was increased in temporal tendons and masseter aponeurosis following exposure to tensile strain. Moreover, the expression of tubulin beta 3 class III, which promotes cell cycle progression, and septin 9, which promotes cytoskeletal rearrangements, were decreased in stretched Achilles tendon cells and their expression was increased in stretched masseter aponeurosis and temporal tendon cells. In conclusion, cyclic strain differentially affects gene expression in Achilles tendons and tendons of the masticatory muscles.

Cognitive skills and the underlying neural architecture are under the influence of genetics. Cognitive genomics research explores the triadic relationship between genes, brain, and cognition, with its major strategy being genotype-driven. Here we show that an inverse strategy is feasible to identify novel candidate genes for particular neuro-cognitive phenotypes in macaques. Two monkeys, originally involved in separate psychological studies, exhibited learning delay and low levels of social performance monitoring. In one monkey, mirror neurons were fewer compared to controls and mu suppression was absent in the frontal cortex. The other monkey showed heightened visual responsiveness in both frontal cortex and dopamine-rich midbrain, with a lack of inter-areal synchronization. Exome analyses revealed that the two monkeys were most likely cousins and shared variants in MAP2, APOC1, and potentially HTR2C. This phenotype-driven strategy in cognitive genomics provides a useful means to clarify the genetic basis of phenotypic variation and develop macaque models of neuropsychiatric disorders.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

The serotonin 5-HT2A receptor (5-HT2AR) has been receiving increasing attention because its genetic variants have been associated with a variety of neurological diseases. To elucidate the pathogenesis of the neurological diseases associated with 5-HT2AR gene (HTR2A) variants, we have previously established a protocol to induce HTR2A-expressing neurons from human-induced pluripotent stem cells (hiPSCs). Here, we investigated the maturation stages and electrophysiological properties of HTR2A-positive neurons induced from hiPSCs and constructed an HTR2A promoter-specific reporter lentivirus to label the neurons. We found that neuronal maturity increased over time and that HTR2A expression was induced at the late stage of neuronal maturation. Furthermore, we demonstrated successful labelling of the HTR2A-positive neurons, which had fluorescence and generated repetitive action potentials in response to depolarizing currents and an inward current during the application of TCB-2, a selective agonist of 5-HT2ARs, respectively. These results indicated that our in vitro model mimicked the in vivo dynamics of 5-HT2AR. Therefore, in vitro monitoring of the function of HTR2A-positive neurons induced from hiPSCs could help elucidate the pathophysiological mechanisms of neurological diseases associated with genetic variations of the HTR2A gene.

<jats:p>Video-based markerless motion capture permits quantification of an animal's pose and motion, with a high spatiotemporal resolution in a naturalistic context, and is a powerful tool for analyzing the relationship between the animal's behaviors and its brain functions. Macaque monkeys are excellent non-human primate models, especially for studying neuroscience. Due to the lack of a dataset allowing training of a deep neural network for the macaque's markerless motion capture in the naturalistic context, it has been challenging to apply this technology for macaques-based studies. In this study, we created MacaquePose, a novel open dataset with manually labeled body part positions (keypoints) for macaques in naturalistic scenes, consisting of &amp;gt;13,000 images. We also validated the application of the dataset by training and evaluating an artificial neural network with the dataset. The results indicated that the keypoint estimation performance of the trained network was close to that of a human-level. The dataset will be instrumental to train/test the neural networks for markerless motion capture of the macaques and developments of the algorithms for the networks, contributing establishment of an innovative platform for behavior analysis for non-human primates for neuroscience and medicine, as well as other fields using macaques as a model organism.</jats:p>

Individual cell shape and integrity must precisely be orchestrated during morphogenesis. Here, we determine function of type II cadherins, Cdh6, Cdh8, and Cdh11, whose expression combinatorially demarcates the mouse neural plate/tube. While CRISPR/Cas9-based single type II cadherin mutants show no obvious phenotype, Cdh6/8 double knockout (DKO) mice develop intermingled forebrain/midbrain compartments as these two cadherins' expression opposes at the nascent boundary. Cdh6/8/11 triple, Cdh6/8 or Cdh8/11 DKO mice further cause exencephaly just within the cranial region where mutated cadherins' expression merges. In the Cdh8/11 DKO midbrain, we observe less-constricted apical actin meshwork, ventrally-directed spreading, and occasional hyperproliferation among dorsal neuroepithelial cells as origins for exencephaly. These results provide rigid evidence that, by conferring distinct adhesive codes to each cell, redundant type II cadherins serve essential and shared roles in compartmentalization and neurulation, both of which proceed under the robust control of the number, positioning, constriction, and fluidity of neuroepithelial cells.

Speciation in the open ocean has long been studied, but it remains largely elusive what factors promote or inhibit speciation in such an open environment. Marine amniotes, which evolved from terrestrial ancestors, provide valuable opportunities for studying speciation in the ocean because of their evident aquatic origins. Sea snakes are phylogenetically related to terrestrial elapid snakes and consist of two monophyletic groups (Hydrophiini and Laticaudini). These two groups migrated from land to water almost at the same time, but species diversities are remarkably different: there are approx. 60 species in 16 genera described for hydrophiins, whereas only eight species in the genus Laticauda are described for laticaudins. Here, we provide a high-quality reference genome assembly of a laticaudin L. colubrina with a scaffold N50 value of 40 Mbp, and focused on laticaudins to consider why they have seldom speciated. We performed whole-genome shotgun sequencing of several species of laticaudins sampled in their southmost (Vanuatu) and northmost (Ryukyu) habitats. Demographic histories of Vanuatu and Ryukyu populations suggest that populations of broadly distributed major species are geographically structured. Each species is genetically clearly distinguished, but there is a considerable amount of gene flow between two sibling species distributed sympatrically in Vanuatu. In addition, inter-species genomic admixture is ubiquitously observed among laticaudins even between phylogenetically distant species. Broad distribution of major species combined with such genetic mixability might have prevented laticaudins from genetic isolation and speciation.

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

Macaque monkeys are an important animal model where invasive investigations can lead to a better understanding of the cortical organization of primates including humans. However, the tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the smaller macaque brain, high spatial, temporal, and angular resolutions combined with high signal-to-noise ratio are required to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enables adaptation of the Human Connectome Project (HCP) image acquisition protocols to the in-vivo macaque brain. In addition, we adapted HCP preprocessing methods to the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provides the necessary high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold accelerations for dMRI and fMRI. Automated FreeSurfer segmentation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI all performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. Analyses of functional connectivity in fMRI revealed high sensitivity as compared with those from publicly shared datasets. Tractography-based connectivity estimates correlated with tracer connectivity similarly to that achieved using ex-vivo dMRI. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available in studies of the human brain.

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

Marine amniotes, a polyphyletic group, provide an excellent opportunity for studying convergent evolution. Their sense of smell tends to degenerate, but this process has not been explored by comparing fully aquatic species with their amphibious relatives in an evolutionary context. Here, we sequenced the genomes of fully aquatic and amphibious sea snakes and identified repertoires of chemosensory receptor genes involved in olfaction. Snakes possess large numbers of the olfactory receptor (OR) genes and the type-2 vomeronasal receptor (V2R) genes, and expression profiling in the olfactory tissues suggests that snakes use the ORs in the main olfactory system (MOS) and the V2Rs in the vomeronasal system (VNS). The number of OR genes has decreased in sea snakes, and fully aquatic species lost MOS which is responsible for detecting airborne odours. By contrast, sea snakes including fully aquatic species retain a number of V2R genes and a well-developed VNS for smelling underwater. This study suggests that the sense of smell also degenerated in sea snakes, particularly in fully aquatic species, but their residual olfactory capability is distinct from that of other fully aquatic amniotes. Amphibious species show an intermediate status between terrestrial and fully aquatic snakes, implying their importance in understanding the process of aquatic adaptation.

Human chromosome 7 has been the focus of many behavioral, genetic, and medical studies because it carries genes related to cancer and neurodevelopment. We examined the evolution of the chromosome 7 homologs, and the 7q31 region in particular, using chromosome painting analyses and 3 paint probes derived from (i) the whole of chimpanzee chromosome VII (wcVII), (ii) human 7q31 (h7q31), and (iii) the chimpanzee homolog VIIq31 (cVIIq31). The wcVII probe was used instead of the whole human chromosome 7 because the chimpanzee contains additional C-bands and revealed large areas of synteny conservation as well as fragmentation across 20 primate species. Analyses focusing specifically on the 7q31 homolog and vicinity revealed considerable conservation across lineages with 2 exceptions. First, the probes verified an insertion of repetitive sequence at VIIq22 in chimpanzees and bonobos and also detected the sequence in most subtelomeres of the African apes. Second, a paracentric inversion with a breakpoint in the cVIIq31 block was found in the common marmoset, confirming earlier studies. Subsequent in silico comparative genome analysis of 17 primate species revealed that VIIq31.1 is more significantly conserved at the sequence level than other regions of chromosome VII, which indicates that its components are likely responsible for critical shared traits across the order, including conditions necessary for proper human development and wellbeing.

Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes. PIPSL was created through a unique mechanism, whereby distinct genes were assembled at the RNA level, and the resulting chimera was then reverse transcribed and integrated into the genome by the L1 retrotransposon. Expression of PIPSL RNA via its transcription start sites (TSSs) has been confirmed in the testes of humans and chimpanzee. Here, we demonstrated that PIPSL RNA is expressed in the testis of the white-handed gibbon. The 5'-end positions of gibbon RNAs were confined to a narrow range upstream of the PIPSL start codon and overlapped with those of orangutan and human, suggesting that PIPSL TSSs are similar among hominoid species. Reporter assays using a luciferase gene and the flanking sequences of human PIPSL showed that an upstream sequence exhibits weak promoter activity in human cells. Our findings suggest that PIPSL might have acquired a promoter at an early stage of hominoid evolution before the divergence of gibbons and ultimately retained similar TSSs in all of the lineages. Moreover, the upstream sequence derived from the phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 5' untranslated region and/or neighboring repetitive sequences in the genome possibly exhibits promoter activity. Furthermore, we observed that a TATA-box-like sequence has emerged by nucleotide substitution in a lineage leading to humans, with this possibly responsible for a broader distribution of the human PIPSL TSSs.

© 2018, The Author(s). The L strain of Japanese quail exhibits a plumage phenotype that is light yellowish in colour. In this study, we identified a nonsense mutation in the premelanosome protein (PMEL) gene showing complete concordance with the yellowish plumage within a pedigree as well as across strains by genetic linkage analysis of an F2 intercross population using approximately 2,000 single nucleotide polymorphisms (SNPs) that were detected by double digest restriction site-associated DNA sequencing (ddRAD-seq). The yellowish plumage was inherited in an autosomal recessive manner, and the causative mutation was located within an 810-kb genomic region of the LGE22C19W28_E50C23 linkage group (LGE22). This region contained the PMEL gene that is required for the normal melanosome morphogenesis and eumelanin deposition. A nonsense mutation that leads to a marked truncation of the deduced protein was found in PMEL of the mutant. The gene expression level of PMEL decreased substantially in the mutant. Genotypes at the site of the nonsense mutation were fully concordant with plumage colour phenotypes in 196 F2 offspring. The nonsense mutation was not found in several quail strains with non-yellowish plumage. Thus, the yellowish plumage may be caused by the reduced eumelanin content in feathers because of the loss of PMEL function.

The precise biological etiology of autism spectrum disorder (ASD) remains unknown. In this study, we investigated the neuropathology of a monkey model of autism Human ABCA13 is the largest ABC transporter protein, with a length of 5058 amino acids and a predicted molecular weight of >450 kDa. However, the function of this protein remains to be elucidated. This protein is thought to be associated with major psychiatric disease. Using this monkey model of autism with an ABCA13 deletion and a mutation of 5HT2c, we neuropathologically investigated the changes in the neuronal formation in the frontal cortex. As a result, the neuronal formation in the cortex was found to be disorganized with regard to the neuronal size and laminal distribution in the ABCA13 deletion monkey. The catecholaminergic and GABAergic neuronal systems, serotoninergic neuronal formation (5HT2c) were also found to be impaired by an immunohistochemical evaluation. This study suggested that ABCA13 deficit induces the impairment of neuronal maturation or migration, and the function of the neuronal network. This protein might thus play a role in the neurodevelopmental function of the central nervous system and the dysfunction of this protein may be a pathophysiological cause of mental disorders including autism.

© 2018 Xu et al. Molecular maps of the human brain alone do not inform us of the features unique to humans. Yet, the identification of these features is important for understanding both the evolution and nature of human cognition. Here, we approached this question by analyzing gene expression and H3K27ac chromatin modification data collected in eight brain regions of humans, chimpanzees, gorillas, a gibbon, and macaques. An analysis of spatial transcriptome trajectories across eight brain regions in four primate species revealed 1851 genes showing human-specific transcriptome differences in one or multiple brain regions, in contrast to 240 chimpanzee-specific differences. More than half of these human-specific differences represented elevated expression of genes enriched in neuronal and astrocytic markers in the human hippocampus, whereas the rest were enriched in microglial markers and displayed human-specific expression in several frontal cortical regions and the cerebellum. An analysis of the predicted regulatory interactions driving these differences revealed the role of transcription factors in species-specific transcriptome changes, and epigenetic modifications were linked to spatial expression differences conserved across species.

Mutations generate genetic variation and are a major driving force of evolution. Therefore, examining mutation rates and modes are essential for understanding the genetic basis of the physiology and evolution of organisms. Here, we aim to identify germline de novo mutations through the whole-genome surveyance of Mendelian inheritance error sites (MIEs), those not inherited through the Mendelian inheritance manner from either of the parents, using ultra-deep whole genome sequences (&gt;150-fold) from a chimpanzee parent-offspring trio. We identified such 889 MIEs and classified them into four categories based on the pattern of inheritance and the sequence read depth: [i] de novo single nucleotide variants (SNVs), [ii] copy number neutral inherited variants, [iii] hemizygous deletion inherited variants, and [iv] de novo copy number variants (CNVs). From de novo SNV candidates, we estimated a germline de novo SNV mutation rate as 1.48 x 10(-8) per site per generation or 0.62 x 10(-9) per site per year. In summary, this study demonstrates the significance of ultra-deep whole genome sequencing not only for the direct estimation of mutation rates but also for discerning various mutation modes including de novo allelic conversion and de novo CNVs by identifying MIEs through the transmission of genomes from parents to offspring.

Interest in the common marmoset (Callithrix jacchus) as a primate model animal has grown recently, in part due to the successful demonstration of transgenic marmosets. However, there is some debate as to the suitability of marmosets, compared to more widely used animal models, such as the macaque monkey and mouse. Especially, the usage of marmoset for animal models of human cognition and mental disorders, is still yet to be fully explored. To examine the prospects of the marmoset model for neuroscience research, the Marmoset Gene Atlas (https://gene-atlas.bminds.brain.riken.jp/) provides a whole brain gene expression atlas in the common marmoset. We employ in situ hybridization (ISH) to systematically analyze gene expression in neonate marmoset brains, which allows us to compare expression with other model animals such as mouse. We anticipate that these data will provide sufficient information to develop tools that enable us to reveal marmoset brain structure, function, cellular and molecular organization for primate brain research.

Changes in gene expression resulting from epigenetic and/or genetic changes play an important role in the evolutionary divergence of phenotypes. To explore how epigenetic and genetic changes are linked during primate evolution, we have compared the genome-wide DNA methylation profiles (methylomes) of humans and chimpanzees, which have a 1.2% DNA sequence divergence, of sperm, the frontal cortices, B cells, and neutrophils. We revealed that species-specific differentially methylated regions (S-DMRs), ranging from several hundred base pairs (bp) to several kilo base pairs (kb), were frequently associated with sequence changes in transcription factor-binding sites and insertions of Alu and SVA retrotransposons. We then generated a reference macaque sperm methylome map and revealed, in sperm, that both human and chimpanzee S-DMRs arose more frequently owing to methylation loss rather than gain. Moreover, we observed that the sperm methylomes contained many more hypomethylated domains (HMDs), ranging from 20 to 500 kb, than did the somatic methylomes. Interestingly, the sperm HMDs changed rapidly during primate evolution; hundreds of sperm HMDs were specific to humans, whereas most somatic HMDs were highly conserved between humans and chimpanzees. Notably, these human-specific sperm HMDs frequently occurred in regions exhibiting copy number variations. Our findings indicate that primate evolution, particularly in the germline, is significantly impacted by reciprocal changes in the genome and epigenome.

Atypical neurodevelopment in autism spectrum disorder is a mystery, defying explanation despite increasing attention. We report on a Japanese macaque that spontaneously exhibited autistic traits, namely, impaired social ability as well as restricted and repetitive behaviors, along with our single-neuron and genomic analyses. Its social ability was measured in a turn-taking task, where two monkeys monitor each other's actions for adaptive behavioral planning. In its brain, the medial frontal neurons responding to others' actions, abundant in the controls, were almost nonexistent. In its genes, whole-exome sequencing and copy number variation analyses identified rare coding variants linked to human neuropsychiatric disorders in 5-hydroxytryptamine (serotonin) receptor 2C (HTR2C) and adenosine triphosphate (ATP)-binding cassette subfamily A13 (ABCA13). This combination of systems neuroscience and cognitive genomics in macaques suggests a new, phenotype-to-genotype approach to studying mental disorders.

New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution.

Bitter taste receptors (TAS2R proteins) allow mammals to detect and avoid ingestion of toxins in food. Thus, TAS2Rs play an important role in food choice and are subject to complex natural selection pressures. In our previous study, we examined nucleotide variation in TAS2R38, a gene expressing bitter taste receptor for phenylthiocarbamide (PTC), in 333 Japanese macaques (Macaca fuscata) from 9 local populations in Japan. We identified a PTC "non-taster" TAS2R38 allele in Japanese macaques that was caused by a loss of the start codon. This PTC non-taster allele was only found in a limited local population (the Kii area), at a frequency of 29%. In this study, we confirmed that this allele was present in only the Kii population by analyzing an additional 264 individuals from eight new populations. Using cellular and behavioral experiments, we found that this allele lost its receptor function for perceiving PTC. The nucleotide sequences of the allele including flanking regions (of about 10 kb) from 23 chromosomes were identical, suggesting that a non-taster allele arose and expanded in the Kii population during the last 13,000 years. Genetic analyses of non-coding regions in Kii individuals and neighboring populations indicated that the high allele frequency in the Kii population could not be explained by demographic history, suggesting that positive selection resulted in a rapid increase in PTC non-tasters in the Kii population. The loss-of-function that occurred at the TAS2R38 locus presumably provided a fitness advantage to Japanese macaques in the Kii population. Because TAS2R38 ligands are often found in plants, this functional change in fitness is perhaps related to feeding habit specificity. These findings should provide valuable insights for elucidating adaptive evolutionary changes with respect to various environments in wild mammals.

In our institute, we have recently found a child Japanese monkey who is characterized by deep wrinkles of the skin and cataract of bilateral eyes. Numbers of analyses were performed to identify symptoms representing different aspects of aging. In this monkey, the cell cycle of fibroblasts at early passage was significantly extended as compared to a normal control. Moreover, both the appearance of senescent cells and the deficiency in DNA repair were observed. Also, pathological examination showed that this monkey has poikiloderma with superficial telangiectasia, and biochemical assay confirmed that levels of HbA1c and urinary hyaluronan were higher than those of other ( child, adult, and aged) monkey groups. Of particular interest was that our MRI analysis revealed expansion of the cerebral sulci and lateral ventricles probably due to shrinkage of the cerebral cortex and the hippocampus. In addition, the conduction velocity of a peripheral sensory but not motor nerve was lower than in adult and child monkeys, and as low as in aged monkeys. However, we could not detect any individual-unique mutations of known genes responsible for major progeroid syndromes. The present results indicate that the monkey suffers from a kind of progeria that is not necessarily typical to human progeroid syndromes.

Genome studies of mammals in the superorder Euarchontoglires (a clade that comprises the orders Primates, Dermoptera, Scandentia, Rodentia, and Lagomorpha) are important for understanding the biological features of humans, particularly studies of medical model animals such as macaques and mice. Furthermore, the dynamic ecoevolutionary signatures of Euarchontoglires genomes may be discovered because many species in this clade are characterized by their successful adaptive radiation to various ecological niches. In this study, we investigated the evolutionary trajectory of bitter taste receptor genes (TAS2Rs) in 28 Euarchontoglires species based on homology searches of 39 whole-genome assemblies. The Euarchontoglires species possessed variable numbers of intact TAS2Rs, which ranged from 16 to 40, and their last common ancestor had at least 26 intact TAS2Rs. The gene tree showed that there have been at least seven lineage-specific events involving massive gene duplications. Gene duplications were particularly evident in the ancestral branches of anthropoids (the anthropoid cluster), which may have promoted the adaptive evolution of anthropoid characteristics, such as a trade-off between olfaction and other senses and the development of herbivorous characteristics. Subsequent whole-gene deletions of anthropoid cluster TAS2Rs in hominoid species suggest ongoing ectopic homologous recombination in the anthropoid cluster. These findings provide insights into the roles of adaptive sensory evolution in various ecological niches and important clues related to the molecular mechanisms that underlie taste diversity in Euarchontoglires mammalian species, including humans.

Background: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples. Results: We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs. Conclusions: We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.

The extraoral presence of taste signal transduction proteins has recently been reported in rodents and humans. Here, we report for the first time the presence of these signal transduction proteins in the caecum of a non-human primate, the common marmoset. Quantitative RT-PCR data on the gene expression of taste signal transduction molecules (gustducin and TRPM5) in common marmosets suggested high expression in the caecum, which was not observed in other non-human primates. Immunohistochemical analysis confirmed the specific presence of gustducin and taste receptors in marmoset caecal cells. These results may relate to the specific feeding behaviour of marmosets, which consume plant exudates, primarily gums.

Changes in gene expression have been proposed to have an important role in the evolutionary changes in phenotypes. Interspecific changes in gene expression can result not only from genetic changes in regulatory regions but also from epigenetic changes in such regions. Here we report the identification of genomic regions showing differences in DNA methylation between humans and chimpanzees (termed S-DMRs for species-specific differentially methylated regions) on chromosomes 21 and 22. These regional methylation differences are frequently associated with genes, including those relevant to a disease, such as Alzheimer's disease, diabetes mellitus or cancer. Methylation differences are often correlated with changes in promoter activity or alternative splicing. Comparative studies including other great ape species provide evidence for the contribution of genetic changes to some of these S-DMRs. Genetic changes responsible for the S-DMRs include gain or loss of CTCF-binding site and changes in CpG density in microsatellite repeats. Our results suggest that DNA methylation changes, often caused by small sequence changes, contribute to transcriptional and phenotypic diversification in hominid evolution.

Chimpanzees (Pan troglodytes) have region-specific difference in dietary repertoires from East to West across tropical Africa. Such differences may result from different genetic backgrounds in addition to cultural variations. We analyzed the sequences of all bitter taste receptor genes (cTAS2Rs) in a total of 59 chimpanzees, including 4 putative subspecies. We identified genetic variations including single-nucleotide variations (SNVs), insertions and deletions (indels), gene-conversion variations, and copy-number variations (CNVs) in cTAS2Rs. Approximately two-thirds of all cTAS2R haplotypes in the amino acid sequence were unique to each subspecies. We analyzed the evolutionary backgrounds of natural selection behind such diversification. Our previous study concluded that diversification of cTAS2Rs in western chimpanzees (P. t. verus) may have resulted from balancing selection. In contrast, the present study found that purifying selection dominates as the evolutionary form of diversification of the so-called human cluster of cTAS2Rs in eastern chimpanzees (P. t. schweinfurthii) and that the other cTAS2Rs were under no obvious selection as a whole. Such marked diversification of cTAS2Rs with different evolutionary backgrounds among subspecies of chimpanzees probably reflects their subspecies-specific dietary repertoires.

In mammals, bitter taste is mediated by T2R genes, which belong to the large family of seven transmembrane G protein coupled receptors. Because T2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species&apos; specific diets during mammalian evolution. Here, we investigated the sequences of all 28 putative functional chimpanzee T2R genes (cT2Rs) in 46 western chimpanzees to compare the intraspecies variations in chimpanzees to those already known for all 25 human functional T2R genes (hT2Rs). The numbers of functional genes varied among individuals in western chimpanzees, and most chimpanzees had two or three more functional genes than humans. Similarly to hT2Rs, cT2Rs showed high nucleotide diversity along with a large number of amino acid substitutions. Comparison of the nucleotide substitution patterns in cT2Rs with those in five cT2R pseudogenes and 14 autosomal intergenic noncoding regions among the same individuals revealed that the evolution of cT2R genes was almost identical to that of putative neutral regions with slight but significantly positive Tajima&apos;s D values, suggesting that selective constraint on these genes was relaxed with weak balancing selection. These trends have resulted in the occurrence of various divergent alleles of T2Rs within the western chimpanzee populations and in heterozygous individuals who might have the ability to taste a broader range of substances.

Bitter taste perception evolved as a key detection mechanism against the ingestion of bioactive substances, and is mediated by TAS2R gene family members in vertebrates. The most widely known and best studied bitter substance is phenylthiocarbamide (PTC), which is recognized by TAS2R38 and has a molecular structure similar to that of glucosinolates contained in Brassica plants. The "non-taster" phenotypic polymorphism (i.e., not sensitive to PTC-containing foods) has been identified in many primates, including humans. Here, we report genetic and behavioral evidence for the existence of "non-taster" Japanese macaques, which originated from a restricted region of Japan. Comparison of the sequences of the TAS2R38 gene of 333 Japanese and 55 rhesus macaques suggested that this genotype appeared after the divergence of these two species, independently of the appearance of human and chimpanzee "non-tasters". This finding might give a clue for elucidating the ecological, evolutionary, and neurobiological aspects of bitter taste perception of primates, as related to the plants that they sometimes use as foods in their habitats.