Single-neuron diversity generated by Protocadherin-β cluster in mouse central and peripheral nervous systems

Abstract

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.

Keywords: Pcdh; combinatorial expression; monoallelic; neural circuit; neuronal individuality; protocadherin; single-cell 3′-RACE; single-neuron diversity.

Figure 1

Expression of the Pcdh-β transcripts in the mouse brain. (A) RT-PCR analysis of P21 cerebellum using isoform-specific primers demonstrated that all 22 of the Pcdh-β transcripts were expressed. In situ hybridization analysis with a Pcdh-β highly conserved probe (B) and with a probe specific for β15 (C). Sagittal sections of P21 mouse brain are shown. The distributions of the signals were almost identical, but the intensity of the β15-specific probe at the macroscopic level was weaker than that obtained with the Pcdh-β highly conserved probe, suggesting that the expression of the Pcdh-β transcripts overlapped at the macroscopic level.

Figure 2

In situ hybridization analysis of Pcdh-β in the mouse cerebellum. (A–F) Coronal sections. (A) In situ hybridization with the Pcdh-β highly conserved probe; (B–F) in situ hybridization using probes specific for β3 (B), β15 (C), β16 (D), β19 (E), and β22 (F). (G–M) Comparison of staining with the Pcdh-β highly conserved probe (G,I,K,M) and the β15-specific probe (H,J,L,N) at P10–P150.

Figure 3

Split single-cell 3′-RACE analysis of Pcdh-β in Purkinje cells. Purkinje cells were from the P21 F1 progeny of a cross between the B6 and JF1 strains. Individual Purkinje cells are indicated as #1–1 to #1–39. Samples #1–8, #1–16, #1–24, #1–32, and #1–40 were negative controls that contained no cells. The RNAs in each Purkinje cell were reverse transcribed using an adapter-attached oligo dT primer and a Pcp-2-specific primer set. Each RT sample was divided into three separate tubes and subjected to 3′-RACE (PCR). The three independent PCR products are referred to as 1, 2, and 3. The results for electrophoresis of the second-round 3′-RACE (PCR)-products are shown.

Figure 4

Distribution of the B6 and JF1 alleles as the source of each Pcdh-β gene in individual Purkinje cells. To determine whether the amplified 3′-RACE (PCR) products were from the B6 or JF1 allele, we performed direct sequencing of the products in each of the three tubes from 28 Purkinje cells. The samples that contained an insufficient quantity of product for sequencing are indicated with an X. The amplified Pcp-2 products were not sequenced and are indicated with a closed circle.

Figure 5

3′-terminal diversity of Pcdh-β transcripts within single Purkinje cells. Single-cell 3′-RACE analysis of β22 in Cell #2–10 (Figures 5A,C) and β15 in Cell #2–21 (Figures 5B,D). The position of polyadenylation is indicated by red arrowheads. The β22 transcripts in Cell #2–10 showed three types of 3′-termini. The β15 transcripts in Cell #2–21 showed two types of 3′-termini.

Figure 6

In situ hybridization analysis of Pcdh-β in mouse hippocampus and cerebral cortex. Pcdh-β gene expression in P21 mouse hippocampus at low (A,B,C) and high (D–I) magnification, showing the CA1 and CA3 fields. (A,D,G) β2 to β22 expression using the Pcdh-β highly conserved probe. (B,E,H) β15 expression. (C,F,I) β22 expression. Examination of the cerebral motor cortex (J–M), and cerebral visual cortex (N–Q) with specific probes for β22 (L,M,P,Q), and the Pcdh-β highly conserved probe (J,K,N,O). Most neurons were stained by the Pcdh-β highly conserved probe, but the specific probes stained only subsets of these neurons, suggesting that each member of the Pcdh-β subfamily is expressed differentially in subpopulations of these neurons.

Figure 7

In situ hybridization analysis of Pcdh-β genes in mouse trigeminal and dorsal root ganglia. β2 to β22 expression using the Pcdh-β highly conserved probe on embryonic mouse trigeminal ganglion (A) and dorsal root ganglion (D). (B,E) Specific expression of β15; (C,F) specific expression of β22. Most neurons were stained by the Pcdh-β highly conserved probe, but the specific probes stained only subsets of these neurons, suggesting that each member of the Pcdh-β subfamily is expressed differentially in subpopulations of these neurons.

Figure 8

Differential expression of Pcdh-β genes in GABAergic interneurons and cholinergic neurons. (A–I) Double in situ hybridization analysis of Pcdh-β genes (red: HNPP fluorescence) and GAD67 (green) in sagittal sections of P21 mouse hippocampus. (J–O) Double in situ hybridization analysis of Pcdh-β genes (red: false color of NBT/BCIP) and cholinergic neuron markers (ChAT + VAChT, green) in coronal sections of P21 mouse brain stem facial nucleus. Insets: High-magnification photomicrograph of the outlined area. Arrowheads indicate neurons expressing both Pcdh-β and GAD67 or ChAT + VAChT mRNAs (white or black), and β22 mRNA alone (red), respectively. Most neurons were stained by the Pcdh-β highly conserved probe, but the specific probes stained only subsets of these neurons, suggesting that each member of the Pcdh-β subfamily is expressed differentially in subpopulations of GABAergic interneurons in the hippocampus and cholinergic neurons in the facial nucleus.