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

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.


INTRODUCTION
The central and peripheral nervous systems contain an enormous number of neurons, that have diverse characteristics and unique identities that enable them to generate complex neural circuits. Therefore, elucidating the molecular mechanisms that specify the unique characteristics of individual neurons is important for understanding the overall complexity of the nervous system.
Recent studies reported that two families of cell-cell recognition molecules, Dscam1 in insects and clustered protocadherin (Pcdh) in vertebrates, are promising candidates for specifying the unique characteristics of individual neurons (Zipursky and Sanes, 2010;Yagi, 2012). Dscam1 proteins are single-pass transmembrane domain proteins of the immunoglobulin superfamily that play important roles in neural circuit formation (Hattori et al., 2008;Schmucker and Chen, 2009). The fly Dscam1 gene encodes 19,008 different ectodomain isoforms, which are expressed in a biased stochastic fashion in individual neurons, thereby conferring a unique molecular identity on each Drosophila neuron (Neves et al., 2004). In addition, Hattori et al. (2007) showed that Dscam1 diversity is essential for generating complex neural circuits.
Approximately 100 of the diverse cadherin-superfamily genes are highly expressed in vertebrate brain (Takeichi, 2007;Yagi, 2008), and about 50 of these are encoded by the Pcdh gene clusters, Pcdh-α, Pcdh-β, and Pcdh-γ (Kohmura et al., 1998;Wu and Maniatis, 1999;Sugino et al., 2000;Hirayama and Yagi, 2006;Morishita and Yagi, 2007). Cadherin-superfamily proteins play key roles in the morphogenesis and function of the brain, including the formation and maintenance of the neuroepithelium, neurite extension, migration of neuronal cells, synaptogenesis, and synaptic plasticity (Takeichi, 2007;Suzuki and Takeichi, 2008). Previous studies revealed that the clustered Pcdhs play important roles in neuronal survival, axonal projection, synaptic connectivity, and several brain functions, including learning and memory (Wang et al., 2002b;Fukuda et al., 2008;Hasegawa et al., 2008;Katori et al., 2009). Since the clustered Pcdhs show unique gene regulations and form heteromultimeric Pcdh protein tetramers with homophilic binding specificity, the clustered Pcdhs are considered to be candidates for generating complex neural circuitry by providing molecular codes that determine neuronal individuality in the vertebrate central and peripheral nervous systems (Schreiner and Weiner, 2010;Zipursky and Sanes, 2010;Yagi, 2012).
The Pcdh-α, Pcdh-β, and Pcdh-γ gene clusters are tandemly localted on the mouse 18 chromosome. All isoforms of Pcdh-α, Pcdh-β, and Pcdh-γ genes possess their own promoters and variable exons in each gene cluster in the same direction (Wu et al., 2001). The Pcdh-α or Pcdh-γ variable exons are spliced onto each Pcdh-α or Pcdh-γ constant exons, respectively, that encode the common 3 regions. In contrast, the Pcdh-β gene cluster does not contain constant exons and is instead comprised of unspliced single exon genes.
Extensive investigation of the gene regulation mechanisms in the clustered Pcdhs has identified some unique features. In situ hybridization analysis showed that the Pcdh-α and Pcdh-γ genes are differentially regulated at the level of individual neurons (Kohmura et al., 1998;Esumi et al., 2005;Frank et al., 2005), and single-cell analysis of Purkinje cells using multiplex RT-PCRs established that one or two variable exons in the Pcdh-α and Pcdh-γ clusters are stochastically expressed from each allele in individual Purkinje cells (Esumi et al., 2005;Kaneko et al., 2006). The expression of the clustered Pcdh genes is governed by the activation of individual promoters from among multiple promoters in the cluster (Tasic et al., 2002;Wang et al., 2002a). Furthermore, the discovery of a long-range regulatory element in the Pcdh-α (Ribich et al., 2006;Kehayova et al., 2011) and Pcdh-β (Yokota et al., 2011) gene clusters provided further support for a model in which promoter choice among multiple promoters drives unique expression patterns in single neurons.
To date, little information has been published on the expression of the Pcdh-β genes, which consist of 22 single exon genes located on mouse chromosome 18 (Vanhalst et al., 2001), although the postsynaptic localization of one Pcdh-β protein, Pcdh-β16, was reported for mouse and primate retina (Junghans et al., 2008;Puller and Haverkamp, 2011). Here, we show that the expression patterns of the Pcdh-β genes are similar to those of the Pcdh-α and Pcdh-γ genes. 3 -RACE analysis of single Purkinje cells showed that Pcdh-β isoforms derived from monoallelic chromosomes were combinatorially expressed in individual Purkinje cells. Furthermore, in situ hybridization analysis showed, for the first time, scattered expression patterns at the level of individual neurons in the trigeminal ganglion, dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous findings on the single-neuron diversity generated by Pcdh expression and suggest that Pcdh-β cluster genes may contribute to specifying the identity and diversity of individual neurons.

ANIMALS
C57BL/6 (B6) mice were purchased from Charles River Japan. F1 hybrid offspring were obtained by intercrossing mice of the laboratory strain B6 with the Japanese wild mouse strain JF1, obtained from the National Institute for Genetics (Mishima, Shizuoka, Japan). All procedures undertaken in this study were approved by our institute's Animal Care and Use Committee and conform to Japanese guidelines.

RT-PCR
Total RNA was prepared from P21 B6 mouse cerebellum using Trizol reagent (Invitrogen) and reverse-transcribed by Superscript III reverse transcriptase (Invitrogen), using random primers, according to the manufacturer's protocol. The PCR of each Pcdh-β isoform was performed using cDNA as the template, 2 μl of 10× Ex-taq PCR buffer, 2 μl of 2.5 mM each dNTP mix, 0.4 μl of RT-PCR primer set, 0.1 μl of Ex-taq HS polymerase (Takara, Japan), and water to a final volume of 20 μl. The PCR conditions were 3 min at 94 • C and then 35 cycles of 30 s at 94 • C, 30 s at 60 • C (for Pcdh-β1) or 58 • C (for other Pcdh-β genes), and 2 min at 72 • C. The primer sets and sequences are shown in Table S1.
To detect the FITC-labeled probes, the sections were incubated with an anti-FITC antibody conjugated with horseradish peroxidase (Jackson Immuno Research Laboratory, 1:4000 in the blocking buffer) for 2-5 hrs at room temperature. After being washed in TNT three times for 15 min, the sections were treated with 1:50 diluted TSA-Plus [dinitrophenol (DNP)] reagents for 5 min, according to the manufacturer's instructions (Perkin-Elmer, Wellesley, MA), which converted the FITC signals to DNP signals. After being washed in TNT three times for 10 min, the sections were incubated overnight at 4 • C with an anti-DNP antibody conjugated with Alexa488 (1/500, Molecular Probes) in 1% blocking buffer. At this point, an anti-DIG antibody conjugated with AP (1:1000, Roche Diagnostics) was included in the incubation, to detect the DIG-labeled probes. The AP activity was visualized with NBT/BCIP (see above) or with HNPP fluorescence.
To detect the DIG-labeled probes with HNPP fluorescence, the sections were washed three times in TNT, once in TS 8.0 (0.1 M Tris-HCl, pH 8.0, 0.1 M NaCl, 50 mM MgCl 2 ), and the AP activity was detected using an HNPP fluorescence detection set (Roche Diagnostics), according to the manufacturer's instructions. The incubation for this substrate was carried out for 30 min and stopped by washing in TS 8.0. The sections were then counterstained with 4 ,6-diamidino-2-phenylindope, dihydrochloride (DAPI, Invitrogen, USA) diluted in TS 8.0-100 ng/ml for 3 min. After a brief wash in TS 8.0, the sections were mounted with CC/Mount (Diagnostic Biosystems, Pleasanton, CA) mounting medium.

SPLIT SINGLE-CELL 3 -RACE OF PURKINJE CELLS
A split single-cell 3 -RACE method for single Purkinje cells was developed to analyze the one-exon Pcdh-β3, β9, β10, β15, β19, and β22 genes, and was based on our previously published method for single-cell RT-PCR (Esumi et al., 2006). The primer sets and sequences are shown in Table S1 (for split single-cell 3 -RACE). In brief, Purkinje cells were prepared from the cerebellum of P21 mice, from the F1 litter of a B6 female × JF1 male. The tissue was dissected and digested with 90 units of papain (Worthington) at 37 • C for 30 min in 10 ml of dissociation solution [0.002% DL-cysteine HCl (Sigma), 0.05% DNase I (Sigma), 0.1% bovine serum albumin (Sigma), and 0.05% glucose (Nacalai Tesque)]. The digested tissue was spun for 8 min at 300× g, and the pellet was resuspended in Dulbecco's modified Eagle's medium (Sigma). To remove debris, the cells were filtered through a 100-μm cell strainer (Falcon). Single Purkinje cells were picked up by glass capillary and placed in thin, 200μl PCR tubes, with 6 μl of RNase-free water. Complementary DNA was synthesized from the single-cell samples after adding 12.5 pmol/reaction of adaptor-attached oligo dT primer and 2.5 pmol/reaction of Pcp-2 RT primer in a total volume of 10 μl, using six units of Thermoscript reverse transcriptase (Invitrogen), according to the manufacturer's protocol, at 55 • C for 60 min. The reaction was stopped by heating at 85 • C for 5 min.
The cDNAs derived from a single Purkinje cell were split into three PCR tubes as 3.3-μl aliquots and used as a template for the first round of a multiplex PCR, which was performed using the 3.3 μl of cDNA, 2.5 μl of 10× Ex-taq PCR buffer, 2 μl of 2.5 mM each dNTP mix, 1.5 μl of the first PCR primer set, 0.1 μl of Ex-taq HS polymerase (Takara), and 15.6 μl of water. The PCR conditions were an initial 3 min at 95 • C and then 28 cycles of 20 s at 95 • C, 20 s at 58 • C, and 5 min at 68 • C. The first PCR products were split into 0.5-μl aliquots, which were used as the template in the second round of nested PCR with 2 μl of 10× Ex-taq PCR buffer, 1.6 μl of 2.5 mM each dNTP mix, 0.4 μl of the second PCR primer set, 0.1 μl of Ex-taq HS polymerase (Takara), and 15.4 μl of water. The PCR conditions for the Pcdh-β genes were an initial 3 min at 96 • C, 40 cycles of 30 s at 96 • C, 15 s at 56 • C (for β3, β9, and β15) or 58 • C (for β10, β19, and β22), and 1 min at 72 • C. The PCR conditions for the β-actin and Pcp-2 were described in previous reports (Esumi et al., 2005(Esumi et al., , 2006Kaneko et al., 2006). The primer sets and sequences are shown in Table S1.
The secondary PCR products were divided into two tubes. One PCR-amplified product was analyzed by agarose gel electrophoresis: before loading, the sample was mixed with electrophoresis loading buffer (Takara) and heated to 72 • C for 5 min, to sharpen the electrophoretic bands. The other half of the PCR sample was purified by polyethylene glycol precipitation and sequenced by direct sequencing, using a BigDye™ DNA sequencing kit (version 3.1) (ABI). The data were analyzed on an ABI Prism 3100 or an ABI Prism 3730 Genetic Analyzer. Direct sequencing permitted us to identify the allele from which each purified PCR product was derived. The discrimination of monoallelic expression from biallelic expression was accomplished using SeqScape software (ABI). The sequencing results from each 3 -RACE PCR product were confirmed by performing direct sequencing twice, using two distinct pairs of forward and reverse primers.

EXPRESSION OF THE Pcdh-β TRANSCRIPTS IN MOUSE BRAIN
To examine the expression pattern of each Pcdh-β gene in the brain, we first carried out an RT-PCR analysis of all 22 Pcdhβ gene family exons, using cDNA from the P21 mouse cerebellum ( Figure 1A). This analysis clearly showed that all 22 Pcdh-β gene family exons were transcribed in the cerebellum. Next, we identified the 3 ends of some of the Pcdh-β transcripts by 3 -RACE analysis using RNA from P21 mouse cerebellum (Table S2). No spliced product of Pcdh-β with constant region exons of Pcdh-α or Pcdh-γ was detected by first-round RT-PCR analysis (data not shown). These data re-confirmed that the Pcdh-β genes are transcribed as 3 polyadenylated mRNAs (Vanhalst et al., 2001).
The distribution of the Pcdh-β transcripts in the P21 mouse brain was examined by in situ hybridization (Figures 1B,C). Because the DNA sequence that corresponds to extracellular cadherin domain 4 (EC4)-EC5-EC6 of Pcdh-β12 is highly conserved from β2 to β22 (more than 84% similarity), we used this sequence as a pan-Pcdh-β probe (Pcdh-β highly conserved probe, hereafter) to detect all 21 isoforms of Pcdh-β. We excluded the β1 gene from this study because of its low similarity to β2-β22. The Pcdh-β highly conserved probe hybridized widely in the brain, and prominent signals were observed in the olfactory bulb, hippocampus, and cerebellum. No signal was observed with the sense probe (data not shown). These results are consistent with findings obtained for rat brain (Sago et al., 1995).
To examine the distribution of each member of the Pcdh-β subfamily genes, we performed an in situ hybridization analysis using probes that detected specific Pcdh-β isoforms. Sequences showing low similarity (<65%) with other Pcdh-β gene members 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.
were used as probes. Although the Pcdh-β genes are very similar to each other, the similarity of β3, β15, β16, β19, and β22 is relatively low, and could be reliably discriminated. When examined at low magnification, the signals for β15 ( Figure 1C) and the other 4 Pcdh-β genes (data not shown) hybridized in a pattern that was identical to that obtained using the Pcdh-β highly conserved probe, but with lower signal intensity. These results suggested that the Pcdh-β isoforms, at least β3, β15, β16, β19, and β22, have macroscopically overlapping expression patterns in the mouse brain.

Pcdh-β GENES IN SINGLE PURKINJE CELLS
To investigate the gene regulation of the Pcdh-β cluster, we examined the expression patterns of the Pcdh-β transcripts in cerebellar Purkinje cells, since the regulation of the Pcdh-α, and Pcdh-γ clusters was extensively studied in these cells (Esumi  , 2005;Kaneko et al., 2006). In the P21 cerebellum, the Pcdh-β highly conserved probe intensely stained most Purkinje cells (Figure 2A). In contrast, the specific probes for β3, β15, β16, β19, and β22 stained only a subset of the Purkinje cells (Figures 2B-F), showing that the Pcdh-β isoforms, at least β3, β15, β16, β19, and β22, were expressed in a scattered pattern among the Purkinje cells. Next, we analyzed the age-dependence of the expression patterns of the Pcdh-β genes in Purkinje cells. From P10 to P150, all the Purkinje cells were intensely stained by the Pcdh-β highly conserved probe (Figures 2A,G,I,K,M), whereas the specific probe for β15 stained only a subset of Purkinje cells (Figures 2C,H,J,L,N). Likewise, the specific probes for β3, β16, β19, and β22 stained only subsets of the Purkinje cells, resulting in scattered patterns such as those seen at P21 (data not shown). Therefore, the expression of the β3, β15, β16, β19, and β22 isoforms, and probably all of the Pcdh-β isoforms, was scattered among Purkinje cells from P10 to P150. On the other hand, in granule cells stained signals for both Pcdh-β highly conserved and specific β15 probes were reduced in P90 and P150.
The next step was to confirm the differential expression revealed by the in situ hybridization, and to further investigate whether the gene regulation of Pcdh-β transcripts in single neurons is monoallelic and combinatorial. For this purpose, we modified our split single-cell RT-PCR method. Since the Pcdh-β genes consist of only one exon (Vanhalst et al., 2001), conventional RT-PCR against Pcdh-β exon could amplify not only the Pcdhβ transcripts but also the genomic DNA, leading us to erroneous conclusions. Fortunately, the Pcdh-β transcripts are polyadenylated (Table S2), so we adapted our method to 3 -RACE. First, we searched for a region of strong similarity shared by several of the Pcdh-β genes to use in the first multiplex primer annealing and found that base pairs 2289-2307 of β15 are highly conserved in β3, β9, β10, β15, β19, and β22. We next sequenced the region proximal to the functional polyadenylation signal of these six Pcdh-β genes in the JF1 mouse strain, and detected polymorphisms against the B6 strain ( Figure S1). These data ultimately led us to choose β3, β9, β10, β15, β19, and β22 as the targets for this analysis. We tested our split single-cell 3 -RACE method using Purkinje cells of P21 F1 mice from a B6 × JF1 cross. After optimizing the experimental conditions, we reproducibly obtained the predicted fragments for β3, β9, β10, β15, β19, β22, β-actin (positive control for 3 -RACE amplification), and Pcp-2 (a marker for Purkinje cells). Only single Purkinje cell-derived cDNA samples that showed intense expressions of Pcp-2 and β-actin were used for the expression analysis of the individual Pcdh-β isoforms in subsequent experiments.
Next, we analyzed the expression patterns of the β3, β9, β10, β15, β19, and β22 transcripts in single Purkinje cells using the split single-cell 3 -RACE method (Figure 3). Of the 28 single Purkinje cells analyzed, all were positive for the PCR products of Pcp-2 and β-actin, confirming that they were mature Purkinje cells, and their mRNAs were successfully reverse-transcribed. None of the control samples (#1-8, #1-16, #1-24, #1-32, or #1-40), which did not contain Purkinje cells, yielded PCR products, confirming the reliability of this method.
When the cells were subjected to the Pcdh-β gene-specific second-round PCR, 21 cells were positive for some of the Pcdh-β genes. Of these, 15 showed specific amplification of one of the six Pcdh-β genes investigated in all three (3/3) sample tubes (for example, β10 in Cell #1-1, β15 in Cell #1-2, and β19 in Cell #1-5). In addition to the 3/3 PCR amplifications, five Purkinje cells showed specific PCR amplifications in 2/3 tubes (for example, β19 in Cells #1-3, β3 in Cell #1-15, and β22 in Cell #1-18), and specific PCR amplification was seen in 1/3 tubes for two Purkinje cells (β22 in Cell #1-13 and β9 in Cell #1-38). Obtaining only 2/3 and 1/3 positive tubes may suggest that these cells contained a low amount of the corresponding mRNAs. The remaining seven cells were negative for any of the Pcdh-β genes, probably indicating that other Pcdh-β genes not included in this analysis were expressed by these cells. Finally, more than one Pcdh-β isoform was amplified in 3/3 tubes of three Purkinje cells (β9 and β19 in Cell #1-10, β3 and β22 in Cell #1-12, and β9 and β19 in Cell #1-17). These results strongly suggested that the Pcdh-β genes are expressed in Purkinje cells in a differential and combinatorial manner.
Next, each PCR product was directly sequenced to distinguish among three possible patterns of expression: monoallelic expression of the maternal allele, monoallelic expression of the paternal allele, and biallelic expression. In the case of β-actin, all the PCR products showed a biallelic expression pattern. This was consistent with our previous analysis (Esumi et al., 2005;Kaneko et al., 2006), which was obtained by split single-cell RT-PCR. Thus, these findings confirmed the reliability of the present method. In contrast to β-actin, a monoallelic expression pattern was found for the Pcdh-β genes in 17 of the 18 3/3 3 -RACE-positive products (94%) (Figure 4) as follows: β10 in Cell #1-1, β15 in Cell #1-2, β19 in Cell #1-5, β9 in Cell #1-7, β19 in Cell #1-5, β9 and

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.
β19 in Cell #1-10, β19 in Cell #1-11, β3 and β22 in Cell #1-12, β3 in Cell #1-14, β9 and β19 in Cell #1-17, β15 in Cell #1-22, β15 in Cell #1-26, β10 in Cell #1-28, β19 in Cell #1-39. Biallelic expression was observed only for β9 in Cell #1-36. Statistically, if this assay amplified only a single cDNA molecule, even though biallelic products were present in the PCR tube, an artifactual 3/3 monoallelic expression pattern would theoretically be observed for 25% of the cells (Rhoades et al., 2000). Clearly, at 94% vs. 25%, there was a highly significant difference, which was upheld by a χ-square analysis between monoallelic and biallelic expression (P < 10 −4 ). Of the 17 monoallelically expressed genes, seven were derived from the B6 allele and 10 from the JF1 allele. Thus, no allelic distortion from the paternal or maternal allele was found in this assay. Taken together, these results clearly showed that the Pcdh-β cluster genes are regulated monoallelically and combinatorially in individual Purkinje cells. This gene regulation would provide the molecular basis for the random expression of the Pcdh-β cluster genes, similar to the Pcdh-α and Pcdh-γ clusters.

DIVERSITY OF THE 3 -TERMINUS OF THE Pcdh-β TRANSCRIPTS WITHIN SINGLE PURKINJE CELLS
During the expressional analysis of the Pcdh-β transcripts using single-cell 3 -RACE, we simultaneously analyzed whether the 3 -terminus of each Pcdh-β transcript in single neurons was homogeneous or heterogeneous ( Figure 5). In most individual Purkinje cells, the 3 -terminus of each Pcdh-β transcript was homogeneous, although we observed 3 -terminal heterogeneity in two cells: Pcdh-β22 in Cell #2-10 (Figures 5A,C) and Pcdh-β15 in Cell #2-21 (Figures 5B,D). The β22 transcripts in Cell #2-10 showed three types of 3 -termini, while the β15 transcripts in Cell #2-21 showed two types of 3 -termini. These data indicate that the polyadenylation of each Pcdh-β gene within a single neuron is not always homogeneous, suggesting the existence of transcriptional diversity for each Pcdh-β gene.

SCATTERED EXPRESSION OF Pcdh-β TRANSCRIPTS IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEMS
To extend our knowledge of the neuron types that showed random expression of the Pcdh-β cluster genes, which might correspond to the specification of individual neuron identity, we examined the expression of Pcdh-β genes in a wide range of neurons using in situ hybridization. In the P21 hippocampus, intense signals from the Pcdh-β highly conserved probe were observed in all the pyramidal cell-layer neurons in CA1 and CA3 (Figures 6A,D,G). In contrast, the specific probes for β15 and β22 stained only subsets of these neurons (Figures 6B,E,H,  and C,F,I, respectively). The other specific probes (for β3, β16, and β19) also stained only subsets of these neurons (data not shown). Furthermore, in the P21 cerebral cortex, most neurons showed intense staining for the Pcdh-β highly conserved   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.
probe (see Figures 6J and K for staining in the P21 motor cortex and Figures 6N and O for staining in the P21 visual cortex).
In contrast, the specific probe for β22 stained only a subset of neurons in each of these regions (Figures 6L,M,P,Q), as did the other specific probes (data not shown). These results clearly demonstrated that these Pcdh-β gene isoforms were expressed only in subsets of neurons in the cerebral cortex and of pyramidal neurons in the hippocampal CA1 and CA3 areas, indicating that these Pcdh-β gene isoforms are expressed randomly and differentially in these neurons. We also examined the expression of the Pcdh-β transcripts in the trigeminal and dorsal root ganglia. As in the cerebral cortex and the hippocampus, these peripheral nervous system neurons showed prominent signals from the Pcdh-β highly conserved probe (Figures 7A,D), while distinct subsets were labeled with the β15 and β22 probes (Figures 7B,C,E,F) as well as the β3, β16, and β19 probes (data not shown). These results indicated that each Pcdh-β gene isoform is expressed randomly and differentially in the peripheral nervous system (at least in the trigeminal and dorsal root ganglia).
To examine whether the Pcdh-β cluster genes showed randomscattered expression in GABAergic and cholinergic neurons, we performed double in situ hybridization analysis of the Pcdh-β genes with marker genes for GABAergic and cholinergic neurons. In the hippocampus, GABAergic interneurons constitute an inhibitory network that regulates excitatory neurons (Kullmann, 2011). The GABAergic interneurons were labeled with a GAD67 probe (Figures 8D-F) and showed strong labeling with the Pcdh-β highly conserved probe (Figures 8A,G). Distinct subsets of GABAergic interneurons were labeled with the β16 and β22 probes (Figures 8B,H,C,I). In the brain stem facial nucleus, cholinergic neurons, which were labeled with ChAT + VAChT probes (Figures 8L,M) (Arvidsson et al., 1997), showed prominent labeling by the Pcdh-β highly conserved probe (Figures 8J,N), and distinct subsets were labeled with the β16 probe (Figures 8K,O). Taken together, these results revealed that each member of the Pcdh-β cluster genes is expressed in a scattered pattern in a wide range of neuron types in the central and peripheral nervous systems in mouse.

DISCUSSION
In this study, we extended previous analyses on how Pcdh genes contribute to single-neuron diversity, by examining the expression patterns of the Pcdh-β genes using in situ hybridization analysis and the newly developed single-cell 3 -RACE analysis.
Our results revealed that the Pcdh-β isoforms showed randomlike scattered expression in both the central and the peripheral nervous systems, and that the Pcdh-β genes were regulated monoallelically and combinatorially in single Purkinje cells. We argue that the Pcdh-α, Pcdh-β, and Pcdh-γ gene clusters confer unique molecular identities on individual neurons that could be responsible for appropriate neuronal circuit formation. The newly developed split single-cell 3 -RACE method revealed that a variety of 3 -ends of Pcdh-β mRNAs could be expressed by a single Purkinje cell. There are numerous examples of genes with multiple polyadenylation signals, and the 3 ends of their transcripts sometimes vary with the developmental stage or state of the tissue in which they are expressed (Edwalds-Gilbert et al., 1997). However, it is unclear if the variation in the 3 -ends of mRNAs is constant or changeable within a single cell during a single time-frame. The present study revealed that some of the Pcdh-β genes have more than two functional polyadenylation signals, and that the polyadenylation site can vary in a single cell. For example, in Cell #2-10, three different variations of the 3 end of β22 were detected. Two of the three transcripts utilized the same polyadenylation signal, but the polyadenylation was added on a different nucleotide, while the other transcript utilized a different polyadenylation signal (Figure 5). This finding shows that even within a single cell the polyadenylation site can be variable, and suggests that the polyadenylation of the 3 ends of Pcdh-β transcripts may be tightly or stochastically regulated.
A previous study detected the expression of several members of the Pcdh-β genes in mouse and human brain as a single exonic and 3 -polyadenylated form (Vanhalst et al., 2001). Furthermore, several studies have reported the expression of Pcdh-β transcripts in the central nervous system including rat brain, rat hippocampus, and mouse retina (Sago et al., 1995;Schippert et al., 2009;McGowan et al., 2011). Our results confirm these findings and expand them to show that some, if not all, of the 22 Pcdh-β genes are expressed in the central and peripheral nervous system, including the cerebellum, hippocampus, cerebral cortex, trigeminal ganglion, dorsal root ganglion, and brain stem.
Focusing on the individual Pcdh-β genes, our findings of random-like and combinatorial expression of the individual Pcdh-β genes extensively broaden the potential for Pcdh-driven single-neuron diversity. The random-like scattered expression of individual Pcdh-α and Pcdh-γ genes has been previously observed in olfactory bulb periglomerular neurons, hippocampal neurons, Purkinje cells, dorsal root ganglia neurons, and choroid plexus epithelial cells (Kohmura et al., 1998;Esumi et al., 2005;Frank et al., 2005;Kaneko et al., 2006;Prasad and Weiner, 2011;Lobas et al., 2012). In this study, we observed that individual Pcdh-β genes are expressed with a similar scattered pattern in cerebellar Purkinje cells, hippocampus, cerebral cortex, trigeminal ganglia, dorsal root ganglia, GABAergic interneurons, and cholinergic neurons. This pattern persisted during development, in that the random-like scattered expression of the Pcdh-β genes in cerebellar Purkinje cells was observed from P10 to P150. Furthermore, our data clearly showed that the individual Pcdhβ genes are regulated monoallelically and combinatorially in cerebellar Purkinje cells, similar to the Pcdh-α and Pcdh-γ genes.
Our present results suggest that the individual Pcdh-β genes are differentially expressed among the various neuron types in the central and peripheral nervous systems from the juvenile to adult stages, and imply that the Pcdh-β gene cluster is regulated by similar mechanisms to the Pcdh-α and Pcdh-γ genes. The similar genomic organization, involving multiple promoters with a conserved sequence element (CGCT), long-range enhancer element, and multiple CTCF-binding sites, supports this hypothesis (Wu et al., 2001;Ribich et al., 2006;Handoko et al., 2011;Yokota et al., 2011). To date, we have not found any rules governing the spatial distribution of the Pcdh-β transcripts; however it remains to be elucidated whether or not the pattern is truly random. One intriguing possibility is that the Pcdh-β expressional repertoires in individual neurons determine their target status during neural circuit formation; i.e., pre-and post-synaptic neurons might express the same sets of Pcdh-β genes. The present finding that Pcdh-β genes show a scattered expression pattern on possible pre-and post-synaptic neurons in the hippocampal inhibitory interneuron network (Figures 6D-I and 8B,C,E,F,H,I) supports this idea.
The present study showed that the expression of the Pcdh-β gene cluster is monoallelic and combinatorial in single Purkinje cells. This strongly suggests that individual Purkinje cells express a variable repertoire of some, if not all, of the 22 Pcdh-β genes. In this study, we investigated six of the 22 Pcdh-β genes and showed that at least two types of Pcdh-β per allele can be expressed by a single Purkinje cell (see β9 and β19 in Cell #1-17, and β3 and β9 in Cell #2-22). The variety of Pcdh-β species expressed by a single Purkinje cell can be extrapolated from the present results. In the experiments shown in Figures 3 and 4, among the 28 Purkinje cells analyzed, in 18 of them, one or more clear bands representing the PCR product were obtained from all three split tubes (β10 in Cell #1-1, β15 in Cell #1-2, β19 in Cell #1-5, β9 in Cell #1-7, β9 and β19 in Cell #1-10, β19 in Cell #1-11, β3 and β22 in Cell #1-12, β3 in Cell #1-14, β9 and β19 in Cell #1-17, β15 in Cell #1-22, β15 in Cell #1-26, β10 in Cell #1-28, β9 from biallelic expression in Cell #1-36, β19 in Cell #1-39). Therefore, 0.64 (18/28) Pcdh-β genes were expressed per Purkinje cell. Assuming that there is no distortion of expression frequencies between isoforms, and given that there are 22 genes in the Pcdh-β cluster, we can estimate that 2.3 Pcdh-β genes (0.64 multiplied by 22/6) should be expressed per individual Purkinje cell. Thus, at least two Pcdh-β isoforms would be expressed by any given Purkinje cell. If these assumptions prove correct, the Pcdh-β transcripts could, in theory, produce more than 462 (22 × 21) possible combinations (one Pcdh-β isoform from each allele) in single Purkinje cells.
This scenario could be supported by the following two findings. First, Pcdh-β proteins associate with Pcdh-γ proteins (Han et al., 2010). Second, the Cys-(X)5-Cys (C-X5-C) motif is important for the formation and cell-surface expression of covalently bound cis-tetramers, and the C-X5-C motif in the EC1 domain is completely conserved among all clustered Pcdh proteins in vertebrates (Schreiner and Weiner, 2010;Yagi, 2012). Our group plans to examine the formation of heteromultimeric cis-tetramers that include Pcdh-β proteins and their binding activities.
The importance of highly diverse transmembrane molecules in the formation of neuronal circuits has been shown in Drosophila, where Dscam1 molecules are responsible for the correct patterning of neuronal circuits (Hattori et al., 2007(Hattori et al., , 2008Schmucker and Chen, 2009). We therefore speculate that the requirement for single-neuron diversity in neural circuit formation is conserved from insects to vertebrates, and that the Pcdh cluster is responsible for this role in the vertebrate nervous system (Zipursky and Sanes, 2010;Yagi, 2012). Future studies aimed at testing this hypothesis could shed light on the fundamental mechanisms underlying vertebrate neural circuit formation. FIGURE S1 | Partial sequence, of a region close to the 3-polyadenylated site, of some of the Pcdh-β genes analyzed in this study. We found 27 polymorphisms (indicated by a yellow background) that enabled us to distinguish whether the amplified product was from the B6 or JF1 chromosome. The putative polyadenylation signal sequences are shown in red-underlined letters.