The Japanese Archipelago hosts a diverse group of mammalian species, including subterranean moles, whose migratory and dispersion patterns are believed to have been significantly influenced by environmental fluctuations during the Quaternary period. However, the genetic structure of these species has not been extensively studied using large-scale molecular data. In this study, we explored the population structure of 2 widely distributed mole species in Japan: the Lesser Japanese Mole (Mogera imaizumii), found mainly in eastern Honshu with a few isolated patches in western Honshu, and the Large Japanese Mole (M. wogura), distributed in western Honshu, Shikoku, and Kyushu. We constructed a genome sequence for M. wogura using the Chromium system and conducted a reduced genome representation analysis using multiplexed inter-simple sequence repeat genotyping by sequencing on M. imaizumii and M. wogura. Our findings indicate that M. imaizumii comprises 3 major genetic clusters (ncMim- 1 to 3) that are strongly differentiated (fixation index ranging from 0.376 to 0.478). We discovered genetic connections between populations in the southern Kinki area and isolated populations in western Japan. Mogera wogura consists of 5 main genetic clusters (ncMwo- 1 to 5). Evidence of introgression between 2 genetic clusters (ncMwo-2/ncMwo-3) was found among individuals from the Chugoku area, indicating secondary contact between the 2 differentiated populations. Both species shared a similar population boundary within the Kinki area, which may be associated with current and past geographic barriers. We confirmed that the Kinki region serves as an important site for the diversification of moles, where multiple factors (topographic barriers, interspecific interactions, and/or isolation related to vegetation) may have shaped their population genetic structures.

Analysis of genome-scale evolution has been difficult in large, endangered animals because opportunities to collect high-quality genetic samples are limited. There is a need for novel field-friendly, cost-effective genetic techniques. This study conducted an exome-wide analysis of a total of 42 chimpanzees (Pan troglodytes) across six African regions, providing insights into population discrimination techniques. Wild chimpanzee DNA was extracted noninvasively from collected fecal samples using the lysis-buffer storage method. To target genome-scale regions of host DNA, exome-capture sequencing was performed using cost-effective baits originally designed for humans (closely related to chimpanzees). Multivariate analysis effectively discriminated differences in local populations, aiding in the identification of samples' geographical origins. Exome-wide heterozygosity was negatively correlated significantly with genome-wide nonsynonymous-synonymous substitution ratios, suggesting that mutation loads exist at the local population level. Exome sequences revealed functional diversity and protein-coding gene divergence. Segregating pseudogenes were comprehensively annotated, with many being population-specific and others shared among populations. Focusing on multicopy chemosensory receptor genes, the segregating pseudogenes OR7D4 (an olfactory receptor) and TAS2R42 (a bitter taste receptor) were shared among western and eastern chimpanzees. Overall, our analytical framework offers ecological insights into chimpanzees and may be applicable to other organisms.

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 used 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 eight basic social events. Analyses of social looking demonstrated its potential for investigating adaptive behaviors in a social group. These results suggest that this motion capture system will greatly enhance our ability to analyze primate social behavior.

AIMS/HYPOTHESIS:Diabetic retinopathy is characterised by neuroinflammation that drives neuronal and vascular degenerative pathology, which in many individuals can lead to retinal ischaemia and neovascularisation. Infiltrating macrophages and activated retina-resident microglia have been implicated in the progression of diabetic retinopathy, although the distinct roles of these immune cells remain ill-defined. Our aim was to clarify the distinct roles of macrophages/microglia in the pathogenesis of proliferative ischaemic retinopathies. METHODS:Murine oxygen-induced retinopathy is commonly used as a model of ischaemia-induced proliferative diabetic retinopathy (PDR). We evaluated the phenotype macrophages/microglia by immunostaining, quantitative real-time RT-PCR (qRT-PCR), flow cytometry and scRNA-seq analysis. In clinical imaging studies of diabetic retinopathy, we used optical coherence tomography (OCT) and OCT angiography. RESULTS:Immunostaining, qRT-PCR and flow cytometry showed expression levels of M1-like macrophages/microglia markers (CD80, CD68 and nitric oxide synthase 2) and M2-like macrophages/microglia markers (CD206, CD163 and macrophage scavenger receptor 1) were upregulated in areas of retinal ischaemia and around neo-vessels, respectively. scRNA-seq analysis of the ischaemic retina revealed distinct ischaemia-related clusters of macrophages/microglia that express M1 markers as well as C-C chemokine receptor 2. Inhibition of Rho-kinase (ROCK) suppressed CCL2 expression and reduced CCR2-positive M1-like macrophages/microglia in areas of ischaemia. Furthermore, the area of retinal ischaemia was reduced by suppressing blood macrophage infiltration not only by ROCK inhibitor and monocyte chemoattractant protein-1 antibody but also by GdCl3. Clinical imaging studies of diabetic retinopathy using OCT indicated potential involvement of macrophages/microglia represented by hyperreflective foci in areas of reduced perfusion. CONCLUSIONS/INTERPRETATION:These results collectively indicated that heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation in retinal vascular diseases including diabetic retinopathy. This adds important new information that could provide a basis for a more targeted, cell-specific therapeutic approach to prevent progression to sight-threatening PDR.

AUTS2 mutations often exhibit neurodevelopmental disorders and microcephaly. However, how AUTS2 regulates neuron production and affects brain size remains unclear. Here, we show that AUTS2 cooperates with the Polycomb complex PRC2 to regulate gene expression and cortical neurogenesis. Auts2 mutant mice exhibit reduced division of intermediate progenitor cells (IPCs), leading to decreased neurons and thickness in the upper-layer cortex. Expression of Robo1 is increased in the mutants, which in turn suppresses IPC division. Transcriptome and chromatin profiling experiments show that, in IPCs, AUTS2 primarily represses transcription of genes, including Robo1 . Promoter region of AUTS2 target genes is enriched with H3K27me3, a repressive histone modification, but its levels are reduced in Auts2 mutants. AUTS2 interacts with PRC2, and together, they promote IPC division. These suggest that AUTS2 collaborates with PRC2 to repress gene transcription through H3K27 trimethylation, promoting neuron production. This sheds light on AUTS2 pathophysiological mechanisms in neurogenesis and microcephaly. ### Competing Interest Statement The authors have declared no competing interest.

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.

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 F 1 hybrid songbirds by crossing Taeniopygia guttata with Aidemosyne modesta . These F 1 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 F 1 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 F 1 individuals. Our findings provide insights into the emergence of altered learning capabilities through hybridization, linked to cell type–specific transcriptional changes.

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.

Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. 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. In all four cases, major immune cells, such as T cells (15.1–47.7

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.### Competing Interest StatementThe authors have declared no competing interest.

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.

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.

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.

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.

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.

Abstract Individuality during learned behavior is observed across animal species, yet the biological bases underlying this process remain unclear. We utilized interspecific F1 hybrid songbirds possessing heterozygous genomes from Taeniopygia guttata and T. bichenovii to examine developmental neural mechanisms underlying individuality in vocal learning. When tutoring with songs of both parental species, F1 pupils showed vast individual differences in their acquired songs. Approximately 30% of F1 hybrids selectively learned either song of the two parental species, whereas others developed songs merged with the parental species’ traits. Individuality in song learning was formed due to inherent acoustic biases during vocal babbling. Furthermore, the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei represented individually unique transcriptional signatures associated with individual differences in the song acoustic biases. These results indicate that a genetically predisposed motor bias is the origin of individuality in learning motor skills.

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.

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 >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.

This paper proposes a system for pose estimation on monkeys, in diverse and challenging scenarios, and under complex social interactions by using OpenPose. In comparison to most animals used for research, Monkeys present additional difficulties for pose estimation. Multiple degrees of freedom, unique complex postures, intricated social interactions, among others. Our monkey OpenPose trained model is robust against these difficulties. It achieves similar performance as in human pose estimation models, and it can run in Realtime.