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 protocadherin-␣ (Pcdh-␣) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-␣ genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-␣ gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-␣ isoforms and found that 1) the transcription of specific Pcdh-␣ isoforms correlated significantly with the methylation state of the promoter and the 5 (but not the 3) region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-␣ isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-␣ isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5-regions of the Pcdh-␣C1 and ␣C2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-␣1 to -␣12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5-region of the first exon may help regulate differential Pcdh-␣ transcription and that hypermethylation is sufficient to repress transcription.

The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-β, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-β genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-β genes are expressed in a 3′-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-β probe against a conserved Pcdh-β sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-β genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3′-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-β genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-β genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-β cluster genes contribute to specifying the identity and diversity of individual neurons.

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Miniscrew implants have proven to be effective in providing absolute orthodontic anchorage. However, as self-drilling miniscrew implants have become more popular, a problem has emerged, i.e. root contact, which can lead to perforation and other root injuries. To avoid possible root damage, a surgical guide was fabricated and cone-beam computed tomography (CBCT) was used to incorporate guide tubes drilled in accordance with the planned direction of the implants. Eighteen patients (5 males and 13 females; mean age 23.8 years; minimum 10.7, maximum 45.5) were included in the study. Forty-four self-drilling miniscrew implants (diameter 1.6, and length 8 mm) were placed in interradicular bone using a surgical guide procedure, the majority in the maxillary molar area. To determine the success rates, statistical analysis was undertaken using Fisher's exact probability test. CBCT images of post-surgical self-drilling miniscrew implant placement showed no root contact (0/44). However, based on CBCT evaluation, it was necessary to change the location or angle of 52.3 per cent (23/44) of the guide tubes prior to surgery in order to obtain optimal placement. If orthodontic force could be applied to the screw until completion of orthodontic treatment, screw anchorage was recorded as successful. The total success rate of all miniscrews was 90.9 per cent (40/44). Orthodontic self-drilling miniscrew implants must be inserted carefully, particularly in the case of blind placement, since even guide tubes made on casts frequently require repositioning to avoid the roots of the teeth. The use of surgical guides, fabricated using CBCT images, appears to be a promising technique for placement of orthodontic self-drilling miniscrew implants adjacent to the dental roots and maxillary sinuses.