In the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation's role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

The clustered protocadherins (Pcdhs), Pcdh-␣, -␤, and -␥, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-␣ regulatory element in vitro, affects especially the expression of specific Pcdh-␣ isoforms in vivo. We also identified a Pcdh-␤ cluster control region (CCR) containing six HS sites (HS16, 17, 17, 18, 19, and 20) downstream of the Pcdh-␥ cluster. This CCR comprehensively activates the expression of the Pcdh-␤ gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-␥ isoforms and does not affect Pcdh-␣ expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-␥ cluster to activate the upstream Pcdh-␤ genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-␤ expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

BackgroundThe specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections.ResultsWe show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity.ConclusionsOur results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0326-6) contains supplementary material, which is available to authorized users.

High-pressure in situ x-ray diffraction study on CdS and BP was performed using a diamond anvil cell up to 68 GPa at room temperature. The rocksalt structure type CdS was found to transform to a new high-pressure polymorph at about 56 GPa. X-ray diffraction pattern of the new high-pressure polymorph was successfully indexed by the lowest temperature KCN structure type with the distorted rocksalt structure. Volume change accompanied with the transformation was calculated to be about 0.8%. This small volume change may be responsible to the undetectable change in refractive index of CdS at the transformation. No phase transformation was observed on BP: the zincblende structure is stable up to 68 GPa.

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While environmental and energy issues are increasingly attracting global attention, the world is heading towards a sustainable society in which limited resources are used effectively. In these circumstances, the Toyota group is aiming for the realization of "Zeronize", a term coined to represent the minimizing of negative impacts, and "Maximize", meaning to maximize positive impacts. Our current, and future, tribological innovation of vehicle and process technology to realize these will be described in this report.