Replication‐coupled passive DNA demethylation for the erasure of genome imprints in mice

Genome‐wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication‐coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re‐evaluation of and provide novel insights into the mechanism of genome‐wide DNA demethylation in PGCs.     

Complete genome sequences of rat and mouse segmented filamentous bacteria, a potent inducer of th17 cell differentiation

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E.?coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.     

Loss of heterochromatin protein 1 gamma reduces the number of primordial germ cells via impaired cell cycle progression in mice

Signals from extraembryonic tissues in mice determine which proximal epiblast cells become primordial germ cells (PGCs). After their specification, approximately 40 PGCs appear at the base of the allantoic bud and migrate to the genital ridges, where they expand to about 25?000 cells by Embryonic Day (E)13.5. The heterochromatin protein 1 (HP1) family members HP1alpha, HP1beta, and HP1gamma (CBX5, CBX1, and CBX3, respectively) are thought to induce heterochromatin structure and to regulate gene expression by binding methylated histone H3 lysine 9. We found a dramatic loss of germ cells before meiosis in HP1gamma mutant (HP1gamma(-/-)) mice that we generated previously. The reduction in PGCs in HP1gamma(-/-) embryos was detectable from the early bud stage (E7.25), and the number of HP1gamma(-/-) PGCs was gradually reduced thereafter. Bromodeoxyuridine incorporation into PGCs was significantly reduced in E7.25 and E12.5 HP1gamma(-/-) embryos. Furthermore, a lower proportion of HP1gamma(-/-) PGCs than wild-type PGCs was in S phase, and a higher proportion, respectively, was in G1 phase at E12.5. Moreover, the proportion of p21 (Cip, official symbol CDKN1A)-positive HP1gamma(-/-) PGCs was increased, suggesting that the G1/S phase transition was inhibited. However, no differences were detected between fate determination, migration, apoptosis, or histone modification of PGCs of control embryos and those of HP1gamma(-/-) embryos. Therefore, the reduction in PGCs in HP1gamma(-/-) embryos could be caused by impaired cell cycle in PGCs. These results suggest that HP1gamma plays an important role in keeping enough germ cells by regulating the PGC cell cycle.     

A wide-range phylogenetic analysis of Zic proteins: implications for correlations between protein structure conservation and body plan complexity

We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon-intron boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in a bilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZF-NC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The "conserved" class includes Zic proteins from the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the "diverged" class contains those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.     

Quantitative imaging of fibrotic and morphological changes in liver of non-alcoholic steatohepatitis (NASH) model mice by second harmonic generation (SHG) and auto-fluorescence (AF) imaging using two-photon excitation microscopy (TPEM)

Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively.     

Innervation zones of the upper and lower limb muscles estimated by using multichannel surface EMG

The distribution of innervation zones was investigated in 3 subjects for 17 muscles and 8 muscle groups in the upper and lower limb, by detecting bi-directional propagation of motor unit action potentials (MUAPs) with the multichannel surface electrode array. Clarification of the distribution of innervation zones depended on the ease in detecting the propagation of MUAPs and the actual scattering of innervation zones, which were closely related with muscle morphology with respect to the arrangements of muscle fibers. In muscles having fibers running parallel to each other, such as the biceps brachii, intrinsic hand muscles, vastus lateralis and medialis, tensor fasciae latae, peronei, soleus, tibialis anterior, and hypothenar muscles in the foot, it was relatively easy to detect the propagating MUAPs, and the innervation zones were distributed in a relatively narrow band around muscle belly. On the other hand, in muscles with a complicated structure including pinnation of muscle fibers, in-series muscle fibers and aponeurotic tissues, such as the deltoid, flexors and extensors in the forearm, rectus femoris, sartorius, hamstrings and gastrocnemius, it was more difficult to detect the propagating MUAPs and to identify the innervation zones, which were widely scattered or distributed in complex configurations. The distribution of the innervation zones clarified in the present study can be used to find the optimal location of electrodes in surface EMG recordings and of stimulus electrodes in the functional and therapeutic electrical stimulations. It may also be useful in motor point biopsy for diagnosis of neuromuscular diseases as well as in the botulinum toxin injection for the treatment of spasticity.     

Genetic variation versus recombination rate in a structured population of mice

The correlation between genetic variation and recombination rate was investigated in a structured mouse population. Nucleotide sequence data from 19 autosomal DNA loci from eight inbred strains of mouse (Mus musculus) sampled from three major subspecies were analyzed. The recombination rate was estimated from the comparison of genetic and physical map distances between markers flanking a 10-cM region of each locus. The strains were categorized into four groups (subpopulations) based on geography. By partitioning the genetic diversity into within-group and among-group variation, we detected a positive correlation between the recombination rate and nucleotide diversity within groups. The level of nucleotide differentiation among groups (G(ST)) showed a negative correlation with the rate of recombination. There was no significant correlation between recombination rate and nucleotide diversity when data from different subpopulations were pooled. No correlation was detected between recombination rate and nucleotide divergence of M. musculus and M. spicilegus. These patterns deviate from the strict neutral expectation under the constant nucleotide substitution rate, and they are likely to have been formed either by a hitchhiking effect of positively selected mutants or by background selection of deleterious mutants occurring in a subdivided population. Our series of comparisons show that because a real population always has some structure, incorporation of its information is important in detecting non-neutral evolution.     

Nucleotide sequence comparison of a chromosome rearrangement on human chromosome 12 and the corresponding ape chromosomes

Chromosome rearrangement has been considered to be important in the evolutionary process. Here, we demonstrate the evolutionary relationship of the rearranged human chromosome 12 and the corresponding chromosome XII in apes (chimpanzee, bonobo, gorilla, orangutan, and gibbon) by examining PCR products derived from the breakpoints of inversions and by conducting shotgun sequencing of a gorilla fosmid clone containing the breakpoint and a "duplicated segment" (duplicon). We confirmed that a pair of 23-kb duplicons flank the breakpoints of inversions on the long and short arms of chimpanzee chromosome XII. Although only the 23-kb duplicon on the long arm of chimpanzee chromosome XII and its telomeric flanking sequence are found to be conserved among the hominoids (human, great apes, and gibbons), the duplicon on the short arm of chimpanzee chromosome XII is suggested to be the result of a duplication from that on the long arm. Furthermore, the shotgun sequencing of a gorilla fosmid indicated that the breakpoint on the long arm of the gorilla is located at a different position 1.9 kb from that of chimpanzee. The region is flanked by a sequence homologous to that of human chromosome 6q22. Our findings and sequence analysis suggest a close relationship between segmental duplication and chromosome rearrangement (or breakpoint of inversion) in Hominoidea. The role of the chromosome rearrangement in speciation is also discussed based on our new results.     

Evolution of hominoids and the search for a genetic basis for creating humanness

The phylogenetic relationship of human and apes are reviewed. The history of molecular phylogenetic studies in this field is then discussed, as is the role of natural selection at the molecular level. It is argued that approximately 10,000 genetic changes are responsible for creating human specific phenotypes. A genome-wide comparison is necessary to decipher those changes.     

Comparative Genetics of Functional Trinucleotide Tandem Repeats in Humans and Apes

Several human neurodegenerative disorders are caused by the expansion of polymorphic trinucleotide repeat regions. Many of these loci are functional short tandem repeats (STRs) located in brain-expressed genes, and their study is thus relevant from both a medical and an evolutionary point of view. The aims of our study are to infer the comparative pattern of variation and evolution of this set of loci in order to show species-specific features in this group of STRs and on their potential for expansion (therefore, an insight into evolutionary medicine) and to unravel whether any human-specific feature may be identified in brain-expressed genes involved in human disease. We analyzed the variability of the normal range of seven expanding STR CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and two nonexpanding polymorphic CAG loci (KCNN3 and NCOA3) in humans, chimpanzees, gorillas, and orangutans. The study showed a general conservation of the repetitive tract and of the polymorphism in the four species and high heterogeneity among loci distributions. Humans present slightly larger alleles than the rest of species but a more relevant difference appears in variability levels: Humans are the species with the largest variance, although only for the expanding loci, suggesting a relationship between variability levels and expansion potential. The sequence analysis shows high levels of sequence conservation among species, a lack of correspondence between interruption patterns and variability levels, and signs of conservative selective pressure for some of the STR loci. Only two loci (SCA1 and SCA8) show a human specific distribution, with larger alleles than the rest of species. This could account, at the same time, for a human-specific trait and a predisposition to disease through expansion.This article contains online supplementary material.