Hideaki Abe

Very few animals habitually manufacture and use tools. It has been suggested that advanced tool behaviour co‐evolves with a suite of behavioural, morphological and life history traits. In fact, there are indications for such an adaptive complex in tool‐using crows (genus Corvus species). Here, we sequenced the genomes of two habitually tool‐using and ten non‐tool‐using crow species to search for genomic signatures associated with a tool‐using lifestyle. Using comparative genomic and population genetic approaches, we screened for signals of selection in protein‐coding genes in the tool‐using New Caledonian and Hawaiian crows. While we detected signals of recent selection in New Caledonian crows near genes associated with bill morphology, our data indicate that genetic changes in these two lineages are surprisingly subtle, with little evidence at present for convergence. We explore the biological explanations for these findings, such as the relative roles of gene regulation and protein‐coding changes, as well as the possibility that statistical power to detect selection in recently diverged lineages may have been insufficient. Our study contributes to a growing body of literature aiming to decipher the genetic basis of recently evolved complex behaviour.

The tuatara ( Sphenodon punctatus ), the only living member of the archaic reptilian order Rhynchocephalia (Sphenodontia) once widespread across Gondwana, is an iconic and enigmatic terrestrial vertebrate endemic to New Zealand. A key link to the now extinct stem reptiles from which dinosaurs, modern reptiles, birds and mammals evolved, the tuatara provides exclusive insights into the ancestral amniotes. The tuatara genome, at ∼5 Gbp, is among the largest vertebrate genomes assembled. Analysis of this genome and comparisons to other vertebrates reinforces the uniqueness of the tuatara. Phylogenetic analyses indicate tuatara diverged from the snakes and lizards ∼250 MYA. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Genome sequence analysis identifies expansions of protein, non-protein-coding RNA families, and repeat elements, the latter of which show an extraordinary amalgam of reptilian and mammalian features. Sequencing of this genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. It also provides important insights into both the technical challenges and the cultural obligations associated with genome sequencing.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific cell surface antigen often expressed in glioblastoma and has drawn much attention as a possible therapeutic target. We performed immunohistochemistry on histology sections of surgical specimens taken from 67 cases with glioblastoma, isocitrate dehydrogenase-wild type, and evaluated the morphological characteristics and distribution of the EGFRvIII-positive tumor cells. We then evaluated the localization of EGFRvIII-expression within the tumor and peritumoral areas. EGFRvIII immunopositivity was detected in 15 specimens taken from 13 patients, including two recurrent specimens taken from the same patient at relapse. Immunofluorescence staining demonstrated that EGFRvIII-positive cells were present in cells positive for glial fibrillary acidic protein (GFAP), and some showed astrocytic differentiation with multiple fine processes and others did not shown. The EGFRvIII-positive cells were located in cellular areas of the tumor, but not in the invading zone. In the two recurrent cases, EGFRvIII-positive cells were markedly decreased in one case and retained in the other. With regard to overall survival, univariate analysis indicated that EGFRvIII-expression in patients with glioblastoma was not significantly associated with a favorable outcome. Double-labeling immunofluorescence staining of EGFRvIII and GFAP showed that processes of large, well differentiated, GFAP-positive glia extend to and surround less differentiated, EGFRvIII-positive glial cells in cellular areas of tumor. However, in the tumor periphery, EGFRvIII-positive tumor cells were not observed. This finding suggests that EGFRvIII is involved in tumor proliferation, but that invading glioma cells lose their EGFRvIII expression.

Neuregulin 3 (NRG3) plays a key role in central nervous system development and is a strong candidate for human mental disorders. Thus, genetic variation in NRG3 may have some impact on a variety of phenotypes in non-mammalian vertebrates. Recently, genome-wide screening for short insertions and deletions in chicken (Gallus gallus) genomes has provided useful information about structural variation in functionally important genes. NRG3 is one such gene that has a putative frameshift deletion in exon 2, resulting in premature termination of translation. Our aims were to characterize the structure of chicken NRG3 and to compare expression patterns between NRG3 isoforms. Depending on the presence or absence of the 2-bp deletion in chicken NRG3, 3 breeds (red junglefowl [RJF], Boris Brown [BB], and Hinai-jidori [HJ]) were genotyped using flanking primers. In the commercial breeds (BB and HJ), approximately 45% of individuals had at least one exon 2 allele with the 2-bp deletion, whereas there was no deletion allele in RJF. The lack of a homozygous mutant indicated the existence of duplicated NRG3 segments in the chicken genome. Indeed, highly conserved elements consisting of exon 1, intron 1, exon 2, and part of intron 2 were found in the reference RJF genome, and quantitative PCR detected copy number variation (CNV) between breeds as well as between individuals. The copy number of conserved elements was significantly higher in chicks harboring the 2-bp deletion in exon 2. We identified 7 novel transcript variants using total mRNA isolated from the amygdala. Novel isoforms were found to lack the exon 2 cassette, which probably harbored the premature termination codon. The relative transcription levels of the newly identified isoforms were almost the same between chick groups with and without the 2-bp deletion, while chicks with the deletion showed significant suppression of the expression of previously reported isoforms. A putative frameshift deletion and CNV in chicken NRG3 are structural mutations that occurred before the establishment of commercial chicken lines. Our results further suggest that the putative frameshift deletion in exon 2 may potentially affect the expression level of particular isoforms of chicken NRG3.

Short tandem repeats (STRs) or microsatellites are well-known sequence elements that may change the spacing between transcription factor binding sites (TFBSs) in promoter regions by expansion or contraction of repetitive units. Some of these mutations have the potential to contribute to phenotypic diversity by altering patterns of gene expression. To explore how repetitive sequence motifs within promoters have evolved in avian lineages under mutation-selection balance, more than 400 evolutionary conserved STRs (ecSTRs) were identified in this study by comparing the 2 kb upstream promoter sequences of chicken against those of other birds (turkey, duck, zebra finch, and flycatcher). The rate of conservation was significantly higher in AG dinucleotide repeats than in AC or AT repeats, with the expansion of AG motifs being noticeably constrained in passerines. Analysis of the relative distance between ecSTRs and TFBSs revealed a significantly higher rate of conserved TFBSs in the vicinity of ecSTRs in both chicken-duck and chicken-passerine comparisons. Our comparative study provides a novel insight into which intrinsic factors have influenced the degree of constraint on repeat expansion/contraction during avian promoter evolution.

Eukaryotic promoters are regions containing various sequence motifs necessary to control gene transcription. Much evidence has emerged showing that structural and/or contextual changes in regulatory elements can critically affect cis-regulatory activity. As sequence motifs can be key factors in maintaining complex promoter architectures, one effective approach to further understand the evolution of promoter regions in vertebrates is to compare the abundance and distribution patterns of sequence motifs in these regions between divergent species. When compared with mammals, the chicken (Gallus gallus) has a very different genome composition and sufficient genomic information to make it a good model for the exploration of promoter structure and evolution. More than 10% of chicken genes contained short tandem repeat (STR) in the region 2 kb upstream of promoters, but the total number of STRs observed in chicken is approximately half of that detected in human promoters. In terms of the STR motif frequencies, chicken promoter regions were more similar to other avian and mammalian promoters than these were to the entire chicken genome. Unlike other STRs, nearly half of the trinucleotide repeats found in promoters partly or entirely overlapped with CpG islands, indicating potential association with nucleosome positions. Moreover, the chicken promoters are abundant with sequence motifs such as poly-A, poly-G and G-quadruplexes, especially in the core region, that are otherwise rare in the genome. Most of sequence motifs showed strong functional enrichment for particular gene ontology (GO) categories, indicating roles in regulation of transcription and gene expression, as well as immune response and cognition. Chicken promoter regions share some, but not all, of the structural features observed in mammalian promoters. The findings presented here provide empirical evidence suggesting that the frequencies and locations of STR motifs have been conserved through promoter evolution in a lineage-specific manner. Correlation analysis between GO categories and sequence motifs suggests motif-specific constraints acting on gene function.

Tonic immobility (TI) is fear-induced freezing that animals may undergo when confronted by a threat. It is principally observed in prey species as defence mechanisms. In our preliminary research, we detected large inter-individual variations in the frequency and duration of freezing behavior among newly hatched domestic chicks (Gallus gallus). In this study we aim to identify the copy number variations (CNVs) in the genome of chicks as genetic candidates that underlie the behavioral plasticity to fearful stimuli. A total of 110 domestic chicks were used for an association study between TI responses and copy number polymorphisms. Array comparative genomic hybridization (aCGH) was conducted between chicks with high and low TI scores using an Agilent 4×180 custom microarray. We specifically focused on 3 genomic regions (>60 Mb) of chromosome 1 where previous quantitative trait loci (QTL) analysis showed significant F-values for fearful responses. ACGH successfully detected short CNVs within the regions overlapping 3 QTL peaks. Eleven of these identified loci were validated by real-time quantitative polymerase chain reaction (qPCR) as copy number polymorphisms. Although there wkas no significant p value in the correlation analysis between TI scores and the relative copy number within each breed, several CNV loci showed significant differences in the relative copy number between 2 breeds of chicken (White Leghorn and Nagoya) which had different quantitative characteristics of fear-induced responses. Our data shows the potential CNVs that may be responsible for innate fear response in domestic chicks.

Polymorphisms in the neurotransmitter-related genes can be a major source of behavioral variations. We searched for polymorphic sites in chicken neurotransmitter-related genes and identified two variable number of tandem repeat (VNTR) loci encompassing the paralog of chicken serotonin transporter gene (5-HTT). Both intronic VNTR were highly polymorphic across chicken breeds and the other Galliformes species, even though predominant alleles were considerably different among breeds. One VNTR locus contained sequences complementary to a conserved motif of CCCTC-binding factor (CTCF) within each repetitive unit, indicating that transcription of chicken 5-HTT paralog may be regulated by the CTCF protein. It is of great interest to contrast these results with previous knowledge on the human 5-HTT that also has CTCF binding sites in the repetitive units of intronic VNTR. Additionally, we measured the degree of impulsiveness in domestic chicks for their preference of immediate/small to large/delayed rewards. A significant difference in the impulsiveness score was detected between two chicken breeds (White Leghorn vs. Boris Brown; P < 0.01), as well as between White Leghorn chicks with different 5-HTT genotypes. These findings imply the possibility that 5-HTT VNTR genotypes may have some impact on chicks' impulsive choice by modifying the serotonergic neurotransmission.