Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation.

In vertebrates that have been examined to date, the sexual identity of germ cells is determined by the sex of gonadal somatic cells. In the teleost fish medaka, a sex-determination gene on the Y chromosome, DMY/dmrt1bY, is expressed in gonadal somatic cells and regulates the sexual identity of germ cells. Here, we report a novel mechanism by which sex chromosomes cell-autonomously confer sexually different characters upon germ cells prior to gonad formation in a genetically sex-determined species. We have identified a novel gene, Sdgc (sex chromosome-dependent differential expression in germ cells), whose transcripts are highly enriched in early XY germ cells. Chimeric analysis revealed that sexually different expression of Sdgc is controlled in a germ cell-autonomous manner by the number of Y chromosomes. Unexpectedly, DMY/dmrt1bY was expressed in germ cells prior to gonad formation, but knockdown and overexpression of DMY/dmrt1bY did not affect Sdgc expression. We also found that XX and XY germ cells isolated before the onset of DMY/dmrt1bY expression in gonadal somatic cells behaved differently in vitro and were affected by Sdgc. Sdgc maps close to the sex-determination locus, and recombination around the two loci appears to be repressed. Our results provide important insights into the acquisition and plasticity of sexual differences at the cellular level even prior to the developmental stage of sex determination.

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates.

DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.

ATF6α/ß-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

ATF6α and ATF6ß are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6ß single-knockout mice develop normally, but ATF6α/ß double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/ß double knockout, but not ATF6α- or ATF6ß single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6ß-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/ß double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/ß-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

CSF1R mutations identified in three families with autosomal dominantly inherited leukoencephalopathy.

Genetic and phenotypic heterogeneities are considerably high in adult-onset leukoencephalopathy, in which comprehensive mutational analyses of the candidate genes by conventional methods are too laborious. We applied exome sequencing to conduct a comprehensive mutational analysis of genes for autosomal dominant leukoencephalopathies. Genomic DNA samples from four patients of three families with autosomal dominantly inherited adult-onset leukodystrophy were subjected to exome sequencing. On the basis of the results, 21 patients with adult-onset sporadic leukodystrophy and one patient with pathologically proven HDLS were additionally screened for CSF1R mutations. Exome sequencing identified heterozygous CSF1R mutations (p.I794T and p.R777W) in two families. I794T has recently been reported as a causative mutation for hereditary diffuse leukoencephalopathy with spheroids (HDLS), and R777W is a novel mutation. Although mutational analysis of CSF1R in 21 sporadic cases revealed no mutations, another novel CSF1R mutation, p.C653Y, was identified in one patient with autopsy-proven HDSL. These variants were located in the PTK domain where the causative mutations cluster. Functional prediction of the mutant CSF1R as well as cross-species conservation of the affected amino acids supports the notion that these variants are pathogenic for HDLS. Exome sequencing is useful for a comprehensive mutational analysis of causative genes for hereditary leukoencephalopathies, and CSF1R should be considered a candidate gene for patients with autosomal dominant leukoencephalopathies.

Extremely slow rate of evolution in the HOX cluster revealed by comparison between Tanzanian and Indonesian coelacanths.

Coelacanths are known as "living fossils" because their morphology has changed very little from that in the fossil record. To elucidate why coelacanths have evolved so slowly is thus of primary importance in evolutionary biology. In the present study, we determined the entire sequence of the HOX cluster of the Tanzanian coelacanth (Latimeria chalumnae) and compared it with that of the Indonesian coelacanth (L. menadoensis), which was available in the literature. The most intriguing result was the extremely small genetic divergence between the two coelacanths. The synonymous divergence of the HOX coding region between the two coelacanths was estimated to be 0.07%, which is ~11-fold smaller than that of human-chimp. When we applied the estimated divergence time of the two coelacanths of 6 million years ago (MYA) and 30 MYA, which were proposed in independent mitochondrial DNA analyses, the synonymous substitution rate of the coelacanth HOX cluster was estimated to be ~11-fold and 56-fold smaller than that of human-chimp, respectively. Thus, the present study implies that the reduction of the nucleotide substitution rate in coelacanth HOX genes may account for the conservation of coelacanth morphology during evolution.

Genome-wide genetic variations are highly correlated with proximal DNA methylation patterns.

5-methyl-cytosines at CpG sites frequently mutate into thymines, accounting for a large proportion of spontaneous point mutations. The repair system would leave substantial numbers of errors in neighboring regions if the synthesis of erased gaps around deaminated 5-methyl-cytosines is error-prone. Indeed, we identified an unexpected genome-wide role of the CpG methylation state as a major determinant of proximal natural genetic variation. Specifically, 507 Mbp (∼18%) of the human genome was within 10 bp of a CpG site; in these regions, the single nucleotide polymorphism (SNP) rate significantly increased by ∼50% (P < 10(-566) by a two-proportion z-test) if the neighboring CpG sites are methylated. To reconfirm this finding in another vertebrate, we compared six single-base resolution methylomes in two inbred medaka (Oryzias latipes) strains with sufficient genetic divergence (3.4%). We found that the SNP rate also increased by ∼50% (P < 10(-2170)), and the substitution rates in all dinucleotides increased simultaneously (P < 10(-441)) around methylated CpG sites. In the hypomethylated regions, the "CGCG" motif was significantly enriched (P < 10(-680)) and evolutionarily conserved (P = ∼ 0.203%), and slow CpG deamination rather than fast CpG gain was seen, indicating a possible role of CGCG as a candidate cis-element for the hypomethylation state. In regions that were hypermethylated in germline-like tissues but were hypomethylated in somatic liver cells, the SNP rate was significantly smaller than that in hypomethylated regions in both tissues, suggesting a positive selective pressure during DNA methylation reprogramming. This is the first report of findings showing that the CpG methylation state is significantly correlated with the characteristics of evolutionary change in neighboring DNA.

UTGB toolkit for personalized genome browsers.

UNLABELLED: The advent of high-throughput DNA sequencers has increased the pace of collecting enormous amounts of genomic information, yielding billions of nucleotides on a weekly basis. This advance represents an improvement of two orders of magnitude over traditional Sanger sequencers in terms of the number of nucleotides per unit time, allowing even small groups of researchers to obtain huge volumes of genomic data over fairly short period. Consequently, a pressing need exists for the development of personalized genome browsers for analyzing these immense amounts of locally stored data. The UTGB (University of Tokyo Genome Browser) Toolkit is designed to meet three major requirements for personalization of genome browsers: easy installation of the system with minimum efforts, browsing locally stored data and rapid interactive design of web interfaces tailored to individual needs. The UTGB Toolkit is licensed under an open source license. AVAILABILITY: The software is freely available at http://utgenome.org/.

MachiBase: a Drosophila melanogaster 5'-end mRNA transcription database.

MachiBase (http://machibase.gi.k.u-tokyo.ac.jp/) provides a comprehensive and freely accessible resource regarding Drosophila melanogaster 5'-end mRNA transcription at different developmental states, supporting studies on the variabilities of promoter transcriptional activities and gene-expression profiles in the fruitfly. The data were generated in conjunction with the recently developed high-throughput genome sequencer Illumina/Solexa using a newly developed 5'-end mRNA collection method.

UTGB/medaka: genomic resource database for medaka biology.

Medaka (Oryzias latipes) is a small egg-laying freshwater teleost native to East Asia that has become an excellent model system for developmental genetics and evolutionary biology. The draft medaka genome sequence (700 Mb) was reported in June 2007, and its substantial genomic resources have been opened to the public through the University of Tokyo Genome Browser Medaka (UTGB/medaka) database. This database provides basic genomic information, such as predicted genes, expressed sequence tags (ESTs), guanine/cytosine (GC) content, repeats and comparative genomics, as well as unique data resources including (i) 2473 genetic markers and experimentally confirmed PCR primers that amplify these markers, (ii) 142,414 bacterial artificial chromosome (BAC) and 217,344 fosmid end sequences that amount to 15.0- and 11.1-fold clone coverage of the entire genome, respectively, and were used for draft genome assembly, (iii) 16,519,460 single nucleotide polymorphisms (SNPs), and 2 859 905 insertions/deletions detected between two medaka inbred strain genomes and (iv) 841 235 5'-end serial analyses of gene-expression (SAGE) tags that identified 344 266 transcription start sites on the genome. UTGB/medaka is available at: http://medaka.utgenome.org/.

Evaluation of image processing programs for accurate measurement of budding and fission yeast morphology.

To study the cellular functions of gene products, various yeast morphological mutants have been investigated. To describe yeast morphology objectively, we have developed image processing programs for budding and fission yeast. The programs, named CalMorph for budding yeast and F-CalMorph for fission yeast, directly process microscopic images and generate quantitative data about yeast cell shape, nuclear shape and location, and actin distribution. Using CalMorph, we can easily and quickly obtain various quantitative data reproducibly. To study the utility and reliability of CalMorph, we evaluated its data in three ways: (1) The programs extracted three-dimensional bud information from two-dimensional digital images with a low error rate (<1%). (2) The absolute values of the diameters of manufactured fluorescent beads calculated with CalMorph were very close to those given in the manufacturer's data sheet. (3) The programs generated reproducible data consistent with that obtained by hand. Based on these results, we determined that CalMorph could monitor yeast morphological changes accompanied by the progression of the cell cycle. We discuss the potential of the CalMorph series as a novel tool for the analysis of yeast cell morphology.

High-dimensional and large-scale phenotyping of yeast mutants.

One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.

Data mining tools for the Saccharomyces cerevisiae morphological database.

For comprehensive understanding of precise morphological changes resulting from loss-of-function mutagenesis, a large collection of 1,899,247 cell images was assembled from 91,71 micrographs of 4782 budding yeast disruptants of non-lethal genes. All the cell images were processed computationally to measure approximately 500 morphological parameters in individual mutants. We have recently made this morphological quantitative data available to the public through the Saccharomyces cerevisiae Morphological Database (SCMD). Inspecting the significance of morphological discrepancies between the wild type and the mutants is expected to provide clues to uncover genes that are relevant to the biological processes producing a particular morphology. To facilitate such intensive data mining, a suite of new software tools for visualizing parameter value distributions was developed to present mutants with significant changes in easily understandable forms. In addition, for a given group of mutants associated with a particular function, the system automatically identifies a combination of multiple morphological parameters that discriminates a mutant group from others significantly, thereby characterizing the function effectively. These data mining functions are available through the World Wide Web at http://scmd.gi.k.u-tokyo.ac.jp/.

SCMD: Saccharomyces cerevisiae Morphological Database.

To study the global regulation of cell morphology, a number of groups have recently reported genome-wide screening data for yeast mutants with abnormal morphology. Despite the relatively simple ellipsoidal shape of yeast cells, in the past, cell morphology researchers have processed information on cells manually. These time-consuming, entirely subjective tasks motivated us to develop image-processing software that automatically extracts yeast cells from micrographs and processes them to measure key morphological characteristics such as cell size, roundness, bud neck position angle, nuclear DNA localization and actin localization. To date, we have retrieved 960,609 cells from 52,988 micrographs of 2531 mutants using our software, and we have published the results in the Saccharomyces cerevisiae Morphological Database (SCMD), which facilitates the analysis of abnormal cells. Our system provides quantitative data for shapes of the daughter and mother cells, localization of the nuclear DNA and morphology of the actin patches. To search for mutants with similar morphological traits, the system outputs a list of mutants ranked by similarity of average morphological parameters. The SCMD is available at http://yeast. gi.k.u-tokyo.ac.jp/.