Allelic Gene Regulation of Pcdh-α and Pcdh-γ Clusters Involving Both Monoallelic and Biallelic Expression in Single Purkinje Cells

Ryosuke Kaneko,Hiroyuki Kato,Yoshimi Kawamura,Shigeyuki Esumi,Teruyoshi Hirayama,Takahiro Hirabayashi,Takeshi Yagi

doi:10.1074/jbc.m605677200

Ryosuke Kaneko, Hiroyuki Kato + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m605677200

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Abstract

The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-alpha and Pcdh-gamma gene clusters are expressed in all of the vertebrates studied. For the Pcdh-alpha isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation. Here we investigated total allelic gene regulation in the Pcdh-alpha and -gamma clusters, including the C-type variable exons (C1 to C5) and the Pcdh-gammaA and -gammaB variable exons in single Purkinje cells. Using split single-cell reverse transcription-PCR analysis, almost all of the Purkinje cells at postnatal day 21 biallelically expressed all the C-type isoforms, whereas the Pcdh-alpha isoforms showed both monoallelic and combinatorial expression. The Pcdh-gammaA and -gammaB isoforms also showed differential regulation in each cell with both monoallelic and combinatorial gene regulation. These data indicated that different types of allelic gene regulation (monoallelic versus biallelic) occurred in the Pcdh-alpha and -gamma clusters, although they were spliced into the same constant exons. It has been reported that each C-type Pcdh-alpha or -gamma transcript has a different expression pattern during brain development, suggesting that the different C-type variable exons may code temporal diversity, although the Pcdh-alpha, -gammaA, and -gammaB isoforms were differential and combinatorial gene regulation within a single cell. Thus, the multiple gene regulations in the Pcdh-alpha and -gamma clusters had a potential mechanism for increasing the diversity of individual neurons in the brain.

Highlights

Nism for specifying the identity and diversity of single neurons is important for realizing the complexity of the brain
The differential expression patterns of the Pcdh-␣ isoforms in single neurons suggested a mechanism for providing the identity and specificity of single neurons in the brain [13]
Single Purkinje cells were prepared from the cerebellum of the F1 mice from B6 female ϫ JF1 male, at P21

Summary

The abbreviations used are

Allelic Gene Regulation in Pcdh-␣ and -␥ Clusters accession number NM_138662), as templates to synthesize digoxigenin-11-UTP-labeled (Roche) cRNA probes. Single Purkinje cells were prepared from the cerebellum of the F1 mice from B6 female ϫ JF1 male, at P21. The tissue was dissected and subjected to enzymatic digestion at 37 °C for 30 min in 10 ml of dissociation solution consisting of 90 units of papain (Worthington), 0.002%. B–E, in situ hybridization was performed on sagittal sections of the mouse cerebellum using digoxigenin-labeled RNA probes of Pcdh-␣ transcripts. Complementary DNA was synthesized from single-cell samples after being primed with 40 coexist for each Pcdh gene cluster.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION