Abstract

Protocadherins 11X and 11Y are cell adhesion molecules of the δ1-protocadherin family. Pcdh11X is present throughout the mammalian radiation; however, 6 million years ago (MYA), a reduplicative translocation of the Xq21.3 block onto what is now human Yp11 created the Homo sapiens-specific PCDH11Y. Therefore, modern human females express PCDH11X whereas males express both PCDH11X and PCDH11Y. PCDH11X/Y has been subject to accelerated evolution resulting in human-specific changes to both proteins, most notably 2 cysteine substitutions in the PCDH11X ectodomain that may alter binding characteristics. The PCDH11X/Y gene pair is postulated to be critical to aspects of human brain evolution related to the neural correlates of language. Therefore, we raised antibodies to investigate the temporal and spatial expression of PCDH11X/Y in cortical and sub-cortical areas of the human fetal brain between 12 and 34 postconceptional weeks. We then used the antibodies to determine if this expression was consistent in a series of adult brains. PCDH11X/Y immunoreactivity was detectable at all developmental stages. Strong expression was detected in the fetal neocortex, ganglionic eminences, cerebellum, and inferior olive. In the adult brain, the cerebral cortex, hippocampal formation, and cerebellum were strongly immunoreactive, with expression also detectable in the brainstem.

Introduction

Many animals use complicated forms of communication, yet the infinitely generative nature of human language is unique to, and perhaps characteristic of, Homo sapiens (Hauser et al. 2002; Chance and Crow 2007). Therefore, one approach to the genetics of language is to search for genes that have been added to the human genome following the split from the chimpanzee lineage.

One candidate, the protocadherin 11X/Y (PCDH11X/Y) gene pair (Crow 2002; Priddle and Crow 2009; Priddle et al. 2010), arose 6 million years ago (MYA) by a reduplicative transposition from the X chromosome (Williams et al. 2006) on to what is now the human Y chromosome. Pcdh11X is present on the X chromosome throughout the mammalian radiation (Kalmady and Venkatasubramanian 2009); however, because of this translocation PCDH11X/Y is now X/Y homologous in humans and in no other extant mammal (Wilson et al. 2006). The case for a sex chromosomal locus for a gene related to language and its functional brain asymmetry is strengthened by observations of the neuropsychological deficits presented by individuals with sex chromosome aneuploidies. Klinefelter's (47,XXY) and triple X syndrome (47,XXX) individuals have delays in language acquisition (Visootsak and Graham 2006; Otter et al. 2010) and Turner's syndrome (45,X) patients have difficulties with spatial tasks (Kesler et al. 2004; Rae et al. 2004). These deficits correlate with the structural (Itti et al. 2006; Rezaie et al. 2008) and functional (Murphy et al. 1997; Itti et al. 2003) brain changes.

Members of the protocadherin family, to which the PCDH11X/Y gene pair belongs, are transmembrane cell adhesion molecules expressed predominantly in the brain (Frank and Kemler 2002) that make up the largest cadherin superfamily (Nollet et al. 2000; Hulpiau and van Roy 2009). PCDHs are classified into α, β, and γ sub-families on the basis of their clustered genetic organization (Wu and Maniatis 1999). An additional non-clustered group, termed δ-PCDHs, can be further subdivided, based on the number of cadherin repeats (ECs) and features of the cytoplasmic domain, into δ1- (the group containing PCDH11X/Y) and δ2-PCDHs (Redies et al. 2005; Vanhalst et al. 2005). Classical cadherins, as a class, are involved in the morphogenesis of diverse tissues through calcium-dependent homophilic cell adhesion mediated by a conserved motif in EC1 of the ectodomain (Gumbiner 2005). By contrast, this motif is absent in the PCDHs, thought to be less involved in the strength of cell–cell connections and more in specificity (Morishita and Yagi 2007). The δ1-family member NF protocadherin is required for the formation of the neural tube in Xenopus (Rashid et al. 2006), and the δ2-family member Pcdh19 is required for the correct neurulation of the forebrain in zebrafish (Emond et al. 2009) via an interaction with N-cadherin (Biswas et al. 2010). γ-Pcdhs are required for synaptic development in the mouse spinal cord and are thought to affect the maintenance or maturation of synapses (Weiner et al. 2005).

The PCDH11X/Y gene pair encodes 2 proteins each comprising an ectodomain of 7 ECs, a short transmembrane region, and a variable length cytoplasmic domain differing between isoforms. Following the translocation, PCDH11X/Y has undergone accelerated evolution in the human lineage (Williams et al. 2006). In the longest isoforms, there have been 5 human-specific changes to the PCDH11X ectodomain and 1 change in the cytoplasmic domain; PCDH11Y has accumulated 17 changes, 7 in the ectodomain, and 10 in the cytodomain (Williams et al. 2006). Three of the PCDH11X ectodomain changes are clustered within EC5: 3D homology modeling predicts that they are mapped closely to one another in space (Priddle et al. 2010). One change, Cys517, is located on the surface of the ectodomain, unpaired to any other cysteine residue and free to form a disulfide bond. Another cysteine (Cys680) is introduced between EC6 and EC7. Both these novel interaction sites may alter the binding characteristics of human PCDH11X through the formation of disulfide bonds, a mechanism previously described (Chen et al. 2007) for the Xenopus δ2-family member paraxial protocadherin, and γ-Pcdh-A3 tetramers (Schreiner and Weiner 2010). The cytoplasmic domain of PCDH11X/Y has been shown to interact with β-catenin and induces the Wnt signaling pathway in cultured prostate cancer cells (Yang et al. 2005). The cytoplasmic domain also contains a protein phosphatase 1α (PP1α)-binding motif, designated CM3, a defining characteristic of the δ1-PCDHs (Vanhalst et al. 2005).

PCDH11X/Y and Disease

Several SNPs in the ectodomain (Giouzeli et al. 2004) and cytoplasmic domain of PCDH11X (Giouzeli et al. 2004; Lopes et al. 2004) have been identified. Although SNPs causing coding changes in the cytoplasmic domain of PCDH11Y have been described (Giouzeli et al. 2004; Lopes et al. 2004; Durand et al. 2005), it is suggested that some of these may be X–Y paralogous sequence variants (Trombetta et al. 2010). No PCDH11X/Y sequence variation has been associated with schizophrenia, autism, bipolar disorder, or attention deficit disorder (Giouzeli et al. 2004; Durand et al. 2005). An intronic SNP in PCDH11X was reported in association with late onset Alzheimer's disease in women (Carrasquillo et al. 2009), but the association was not observed in subsequent studies (Beecham et al. 2010; Lescai et al. 2010; Wu et al. 2010; Miar et al. 2011).

PCDH11X/Y, Language, and Intellectual Function

Independent intragenic deletions in both Xq21.3 and Yp11 involving PCDH11X and PCDH11Y have been identified in a single case of a male child with a severe language delay (Speevak and Farrell 2011). The PCDH11X deletion was inherited from the (phenotypically normal) mother, but the PCDH11Y deletion was not present in the father and therefore appears to be a de novo occurrence. The authors postulate that the deletions interfere with the normal splicing, altering gene expression to disrupt the development of language. In another study (Whibley et al. 2010), 2 brothers with intellectual disability were identified with a 182-kb duplication within intron 2 of PCDH11X, although their mildly affected sister was found not to carry the duplication. One interpretation of these findings is that an interruption of PCDH11X is less well tolerated in males than in females, and this line of thinking has been suggested as a reason for the male propensity to autism and attention deficit hyperactivity disorder (Kopsida et al. 2009). Dibbens et al. (2008) invoked a related mechanism whereby PCDH11Y protects males from epilepsy and mental retardation limited to females associated with mutations of the X (only)-linked PCDH19. Both proposed mechanisms assume the presence of PCDH11Y in human males means that PCDH11X is no longer subject to “meiotic suppression of unsynapsed chromatin” (Turner 2007) and has an inactivation status that differs from that of Pcdh11X in all other animals. However, the inactivation status of PCDH11X/Y remains inconclusive: CpG islands in the promoters of both PCDH11X/Y alleles are unmethylated in male and female controls and all alleles present in Klinefelter's (47, XXY) syndrome are also unmethylated (Ross et al. 2006). In females, both alleles of PCDH11X are unmethylated and expression levels of PCDH11X are twice that of males (Lopes et al. 2006). These findings are consistent with the “escape from X-inactivation” that is held to be characteristic of genes on the X with a homolog on the Y, yet observations of the replication timing of both PCDH11X and PCDH11Y do not support this, suggesting complexity in the relevant epigenetic mechanisms (Wilson et al. 2007). The methylation status of PCDH11X/Y in psychiatric populations could be relevant to these conditions (Crow 2008; Isles and Wilkinson 2008).

Previous studies of the expression of PCDH11X/Y in humans have used the reverse transcription-polymerase chain reaction (RT-PCR) (Blanco et al. 2000; Blanco-Arias et al. 2004) and northern blotting (Yoshida and Sugano 1999), but have been limited to a few broad neuronal areas. Real-time PCR has demonstrated twice as much PCDH11X mRNA in adult female temporal lobes as in males (Lopes et al. 2006). A longitudinal study of the prefrontal cortex (Weickert et al. 2009) has shown that levels of PCDH11X/Y are highest in male neonates, decrease through childhood, and are lowest in adults of both sexes. Expression of PCDH11X/Y in fetal cortex, ganglionic eminence, hippocampal formation, and putamen and caudate, but limited to a few cases, was reported from a study with a major focus on PCDH19 (Dibbens et al. 2008).

A polyclonal antibody was raised against PCDH11X/Y for western blotting and immunoprecipitation of cultured prostate cells (Chen et al. 2002) but thus far, no immunohistochemical studies have addressed PCDH11X/Y expression in the human brain.

The aim of this study was to map the expression of PCDH11X/Y in a series of fetal and adult human brains, using antibodies raised in the absence of commercial products. While the study was under way, a commercial antibody against PCDH11X/Y became available and was subsequently included for comparison.

Materials and Methods

This study was conducted with the approval of the Oxfordshire Clinical Research Ethics Committee. Tissue blocks were taken from 12 fetal and 12 adult brains as detailed in Table 1. A full description of the areas used is provided in Supplementary Tables 1 (fetal) and 2 (adult).

Table 1

Specimens used

Case Sex Age 
Fetal 1 Male 12 PCW 
Fetal 2 Female 13 PCW 
Fetal 3 Female 14 PCW 
Fetal 4 Male 16 PCW 
Fetal 5 Female 18 PCW 
Fetal 6 Female 19 PCW 
Fetal 7 Female 21 PCW 
Fetal 8 Female 24 PCW 
Fetal 9 Female 24 PCW 
Fetal 10 Male 26 PCW 
Fetal 11 Male 27 PCW 
Fetal 12 34 PCW 
Adult 1 Male 49 years 
Adult 2 Male 54 years 
Adult 3 Male 67 years 
Adult 4 Male 66 years 
Adult 5 Female 53 years 
Adult 6 Female 82 years 
Adult 7 Male 68 years 
Adult 8 Female 72 years 
Adult 9 Female 80 years 
Adult 10 Female 67 years 
Adult 11 Female 73 years 
Adult 12 Male 43 years 
Case Sex Age 
Fetal 1 Male 12 PCW 
Fetal 2 Female 13 PCW 
Fetal 3 Female 14 PCW 
Fetal 4 Male 16 PCW 
Fetal 5 Female 18 PCW 
Fetal 6 Female 19 PCW 
Fetal 7 Female 21 PCW 
Fetal 8 Female 24 PCW 
Fetal 9 Female 24 PCW 
Fetal 10 Male 26 PCW 
Fetal 11 Male 27 PCW 
Fetal 12 34 PCW 
Adult 1 Male 49 years 
Adult 2 Male 54 years 
Adult 3 Male 67 years 
Adult 4 Male 66 years 
Adult 5 Female 53 years 
Adult 6 Female 82 years 
Adult 7 Male 68 years 
Adult 8 Female 72 years 
Adult 9 Female 80 years 
Adult 10 Female 67 years 
Adult 11 Female 73 years 
Adult 12 Male 43 years 

PCW, postconceptional weeks.

Antibodies

Three antibodies against PCDH11X/Y were raised and used in this study. Procad1a is a mouse monoclonal antibody raised against a synthetic peptide [QEKNYTIREEMPE] corresponding to the N terminus (PCDH11Xa residues 24–36) of all PCDH11X/Y variants. Ex6 is another mouse monoclonal antibody raised against a synthetic peptide [EVPVSVHTRPTDST] corresponding to residues 1023–1037 of the C terminus of PCDH11Ya. X11 is a rabbit polyclonal antibody (made to order, BioCarta, San Diego, CA, United States of America) against a synthetic peptide [LHHSPPLTQATA] corresponding to a consensus sequence from a repeated motif (starting at residue 1158 of PCDH11Xc) within the cytoplasmic region of longer variants of PCDH11X/Y. We also used a commercial rabbit polyclonal antibody raised against a region common to all isoforms of PCDH11X and PCDH11Y (HPA000432, Sigma Aldrich).

Recombinant Proteins

A 357-bp sequence encoding EC1 (119 aa) of PCDH11Xa was directionally cloned into pET24a(+) (69749-3, Merck Chemicals Ltd.). A 333-bp sequence encoding the C terminus (111 aa) of PCDH11Ya was directionally cloned into pGEX-6P-1 (28-9546-48, GE Healthcare UK Ltd.). Large-scale bacterial cultures were grown, expression was induced, and proteins were extracted and purified.

Monoclonal Immunization

Mice were immunized with synthetic peptides, to produce monoclonal antibodies from spleen fusions. Sp2/0 myeloma cells were fused to splenocytes using polyethylene glycol (Harlow and Lane 1988). ClonaCell methylcellulose (03804, StemCell Technologies SARL) was used for cloning and re-cloning of cells following screening against formalin fixed paraffin-embedded human brain tissue. The ability of the antibodies to recognize PCDH11X/Y was assessed by screening with a solid-phase antibody capture enzyme linked immunosorbant assay against recombinant PCDH11Xa EC1 or PCDH11Ya cytodomain. Tissue culture supernatants were removed and diluted 1:5 in phosphate-buffered saline (PBS; 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) and 0.1% sodium azide.

Single-Label Immunohistochemistry

Formalin fixed paraffin-embedded tissues were sectioned at 10 μm. Sections were passed through a series of graded alcohols to remove the paraffin and then microwaved in antigen unmasking solution (H-3300, Vector Laboratories) at low power (without boiling) for 30 min to improve antigen detection (Evers and Uylings 1997). Once cooled, sections were treated with 3% H2O2 for 10 min, then placed into a humidified chamber on a rocking platform (to ensure an even coverage of solutions), and blocked for 90 min in 10% bovine serum and 0.1% Tween-20 diluted in Tris-buffered saline (0.05 M Tris, pH 7.6, 0.15 M sodium chloride).

Primary antibodies were diluted (Procad1a 1:150; Ex6 1:8; X11 1:150; commercial anti-PCDH11X/Y 1:250) in the blocking solution and applied to the tissues for 1 h at room temperature. The humidified chamber was then moved into a refrigerator and the incubation continued overnight (17 h). On the following day, all steps were performed at room temperature on the rocking platform. After a 1-h incubation with biotinylated goat secondary antibodies (anti-mouse 1:200, B 7151; anti-rabbit 1:400, B8895 Sigma Aldrich), the Vectorstain Elite ABC kit (PK-6100, Vector Laboratories) was used to locate the antibody complex. The peroxidase reaction was demonstrated with metal-enhanced 3,3′-diaminobenzidine (DAB, 34065, Perbio Science). The specificity of the immunohistochemical reactions was confirmed by the absence of specific immunoreactivity in control experiments in which the primary antibodies were omitted. Immunoreactivity was inhibited in a dose-dependent manner when Procad1a and Ex6 were incubated with their recombinant proteins and when X11 was incubated with its immunizing peptide prior to their use on tissue sections. A mouse monoclonal isotyping kit (MMT1, AbD Serotec) demonstrated that both Procad1a and Ex6 are immunoglobulins of the IgG1 κ subtype.

Double-Label Immunohistochemistry

Antigen retrieval was performed as for single labeling. Sections were treated with an Avidin/Biotin blocking kit (SP-2001, Vector Laboratories) and then blocked in 10% normal horse serum (NHS) in PBS for 1 h. Primary antibodies were diluted in 2% NHS/PBS at higher concentrations (Procad1a 1:15; Ex6 1:1; X11 1:15; commercial anti-PCDH11X/Y 1:25; doublecortin [DCX] 1:2000, Ab18723 Abcam; neuropeptide Y [NPY] 1:50, Ab10341 Abcam; calbindin D-28k 1:400, #300 Swant; calretinin 1:200, #6B3 Swant; parvalbumin 1:250, #235 Swant) than for single labeling owing to the reduced efficiency of immunofluorescent visualization (Hoffman et al. 2008) and applied to the tissues at 4°C for 17 h, with the exception of the calbindin incubation which lasted 3 days. Biotinylated goat secondary antibodies were diluted (anti-mouse 1:200; anti-rabbit 1:400; anti-guinea pig 1:500, Ab6907 Abcam) in 2% NHS/PBS and applied for 1 h before a 15-min incubation with Fluorescein Avidin distinct cell sorting (DCS) (20 μg/mL in PBS, A-2011, Vector Laboratories). The procedure (minus antigen retrieval) was then repeated for the second primary antibody and visualized with Texas Red Avidin DCS (15 min, 20 μg/mL in PBS, A-2016, Vector Laboratories). Finally, a 5-min incubation with 1% Sudan Black in 70% ethanol was used to reduce lipofuscin like autofluorescence (Schnell et al. 1999) and sections were mounted using Vecta Shield Hard Set (H-1400, Vector Laboratories).

Microscopy

We examined single-labeled sections using a conventional light microscope (Olympus BX50) and light box, and documented results with a digital camera, using the GNU Image Manipulation Program to adjust contrast and brightness on the digitized images. Double-labeled sections were examined using a fluorescent microscope (Nikon Eclipse E600) and Adobe Photoshop CS5 was used to produce merged images.

Results

All 4 PCDH11X/Y antibodies produced a pattern of immunoreactivity, predominantly in the cytoplasm of neurons, which was virtually identical in both groups of brains and will be considered together. Immunoreactivity produced by Procad1a and the commercial antibody directed against all predicted forms of PCDH11X/Y was co-localized within the same cells (Fig. 1A,HI). Furthermore, immunoreactivity produced by Ex6 and X11 directed against the different cytoplasmic domains overlapped in the majority of cells (Fig. 1B,C).

Figure 1.

Double labeling of PCDH11X/Y. (AD) Merged images showing co-localization of all PCDH11X/Y antibodies in the fetal cerebral cortex, 13 PCW female (A, Procad1a: green, anti-PCDH11X/Y: red; B, Procad1a: green, X11: red; C, Ex6: green, X11: red; D, Ex6: green, anti-PCDH11X/Y: red). (E and F) Merged images showing co-localization of PCDH11X/Y with DCX and NPY in the fetal cerebral cortex, 13 PCW female (E, Procad1a: green, DCX: red; F, Procad1a: green, NPY: red). Arrowheads in (E and F) highlight the co-expression of PCDH11X/Y in the SP and IZ with DCX and NPY, respectively. (GK) Adult cerebral cortex, female (G, Procad1a: green; H, anti-PCDH11X/Y: red; I, merged image; J, calretinin: green, anti-PCDH11X/Y: red; K, calbindin: green, anti-PCDH11X/Y: red). Arrowheads in (J and K) highlight the expression of PCDH11X/Y in the absence of calretinin and calbindin, respectively. Scale bars: (AJ) 100 μm; (K): 25 μm. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.

Figure 1.

Double labeling of PCDH11X/Y. (AD) Merged images showing co-localization of all PCDH11X/Y antibodies in the fetal cerebral cortex, 13 PCW female (A, Procad1a: green, anti-PCDH11X/Y: red; B, Procad1a: green, X11: red; C, Ex6: green, X11: red; D, Ex6: green, anti-PCDH11X/Y: red). (E and F) Merged images showing co-localization of PCDH11X/Y with DCX and NPY in the fetal cerebral cortex, 13 PCW female (E, Procad1a: green, DCX: red; F, Procad1a: green, NPY: red). Arrowheads in (E and F) highlight the co-expression of PCDH11X/Y in the SP and IZ with DCX and NPY, respectively. (GK) Adult cerebral cortex, female (G, Procad1a: green; H, anti-PCDH11X/Y: red; I, merged image; J, calretinin: green, anti-PCDH11X/Y: red; K, calbindin: green, anti-PCDH11X/Y: red). Arrowheads in (J and K) highlight the expression of PCDH11X/Y in the absence of calretinin and calbindin, respectively. Scale bars: (AJ) 100 μm; (K): 25 μm. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.

Expression of PCDH11X/Y in the Fetal Human Brain

All PCDH11X/Y antibodies reacted with tissue from all ages, and immunoreactivity was detected in both sexes. The immunoreactivity was strongest in the cortical plate and the ventricular zone of the developing cerebral cortex (Figs 1AF and 2AD), the lateral and medial ganglionic eminences (Fig. 2B), the caudate (Fig. 2A), and the inferior olivary nucleus (Fig. 2G,H). Within the cerebellum, the Purkinje cells and the dentate nucleus were strongly immunoreactive (Fig. 2F). Moderate levels of immunoreactivity were detected in the hippocampal formation (Fig. 2E), the emboliform nucleus of the cerebellum (Fig. 2F), the gracile, cuneate, and spinal trigeminal nuclei (Fig. 2I), the abducens and facial nuclei (Fig. 2F), and the pontine nuclei. Weak immunoreactivity was detected in the subplate (Figs 1AF and 2AC), the thalamus (Fig. 2B), and the arcuate nucleus (Fig. 2H). PCDH11X/Y immunoreactivity was co-localized with DCX in the cortical plate, subplate and intermediate zone (Fig. 1E), and NPY within the subplate, intermediate zone, and subventricular zone (Fig. 1F). A summary of fetal results is shown in Table 2.

Table 2

Regional expression of PCDH11X/Y

Region Fetal Adult 
Frontal cortex CP and VZ Layers II–VI 
 Anterior prefrontal  +++ 
 Cingulate gyrus +++ +++ 
 Motor/premotor +++  
 Posterior orbital gyrus +++ +++ 
 Superior frontal gyrus +++  
 Middle frontal gyrus +++ +++ 
 Inferior frontal gyrus +++  
Temporal cortex CP and VZ Layers II–VI 
 Insular +++  
 Superior temporal gyrus +++ +++ 
 Middle temporal gyrus +++ +++ 
 Inferior temporal gyrus +++  
 Parahippocampal gyrus +++ +++ 
 Temporal pole +++  
Parietal cortex CP and VZ Layers II–VI 
 Postcentral gyrus +++ T.U. 
 Superior lobule +++ T.U. 
 Inferior lobule +++ T.U. 
 Paracentral lobule +++ T.U. 
Occipital cortex CP and VZ Layers II–VI 
 Primary visual  +++ 
Ganglionic eminence  N/A 
 Medial +++  
 Lateral +++  
Hippocampal formation   
 CA1 ++ ++ 
 CA2 ++ ++ 
 CA3 ++ ++ 
 CA4 ++ ++ 
 Fascia dentata ++ ++ 
 Subiculum ++ ++ 
Amygdala T.U.  
 Medial nucleus  
 Central nucleus  
 Basomedial nucleus  
 Basolateral nucleus  
Basal ganglia   
 Caudate +++ ++ 
 Putamen ++ ++ 
Thalamus   
 Anterior nucleus  
 Lateral dorsal nucleus  
 Mediodorsal nucleus 
 Pulvinar nucleus 
Cerebellar cortex   
 Purkinje cells +++ +++ 
 Granule cells 
Cerebellar nuclei   
 Dentate +++ ++ 
 Emboliform ++ ++ 
Midbrain   
 Dorsal raphe 
 Red nucleus T.U. 
 Substantia nigra 
Pons 
Medulla oblongata   
 Abducens nucleus ++ 
 Arcuate nucleus  
 Facial nucleus ++ 
 Hypoglossal nucleus ++ 
 Inferior olivary nucleus +++ ++ 
 Spinal trigeminal nucleus ++ ++ 
 Solitary nucleus T.U. 
Spinal cord T.U. 
Region Fetal Adult 
Frontal cortex CP and VZ Layers II–VI 
 Anterior prefrontal  +++ 
 Cingulate gyrus +++ +++ 
 Motor/premotor +++  
 Posterior orbital gyrus +++ +++ 
 Superior frontal gyrus +++  
 Middle frontal gyrus +++ +++ 
 Inferior frontal gyrus +++  
Temporal cortex CP and VZ Layers II–VI 
 Insular +++  
 Superior temporal gyrus +++ +++ 
 Middle temporal gyrus +++ +++ 
 Inferior temporal gyrus +++  
 Parahippocampal gyrus +++ +++ 
 Temporal pole +++  
Parietal cortex CP and VZ Layers II–VI 
 Postcentral gyrus +++ T.U. 
 Superior lobule +++ T.U. 
 Inferior lobule +++ T.U. 
 Paracentral lobule +++ T.U. 
Occipital cortex CP and VZ Layers II–VI 
 Primary visual  +++ 
Ganglionic eminence  N/A 
 Medial +++  
 Lateral +++  
Hippocampal formation   
 CA1 ++ ++ 
 CA2 ++ ++ 
 CA3 ++ ++ 
 CA4 ++ ++ 
 Fascia dentata ++ ++ 
 Subiculum ++ ++ 
Amygdala T.U.  
 Medial nucleus  
 Central nucleus  
 Basomedial nucleus  
 Basolateral nucleus  
Basal ganglia   
 Caudate +++ ++ 
 Putamen ++ ++ 
Thalamus   
 Anterior nucleus  
 Lateral dorsal nucleus  
 Mediodorsal nucleus 
 Pulvinar nucleus 
Cerebellar cortex   
 Purkinje cells +++ +++ 
 Granule cells 
Cerebellar nuclei   
 Dentate +++ ++ 
 Emboliform ++ ++ 
Midbrain   
 Dorsal raphe 
 Red nucleus T.U. 
 Substantia nigra 
Pons 
Medulla oblongata   
 Abducens nucleus ++ 
 Arcuate nucleus  
 Facial nucleus ++ 
 Hypoglossal nucleus ++ 
 Inferior olivary nucleus +++ ++ 
 Spinal trigeminal nucleus ++ ++ 
 Solitary nucleus T.U. 
Spinal cord T.U. 

CP, cortical plate; N/A, Not applicable; T.U., Tissue unavailable; VZ, ventricular zone.

Figure 2.

Expression of PCDH11X/Y in the fetal brain. Cerebral cortex (A, 14 PCW female; B, 16 PCW male; C, 19 PCW male; D, 12 PCW male), hippocampal formation (E, 18 PCW female), cerebellum and pons (F, 18 PCW female), and medulla oblongata (G, 27 PCW male; H and I, 18 PCW female). Scale bars: (A, FI): 3000 μm; (B, C): 5000 μm; (D) 100 μm; (E) 1000 μm. AN, abducens nucleus; ArN, arcuate nucleus; Cau, caudate; CCtx, cerebellar cortex; CN, cuneate nucleus; CP, cortical plate; DN, dentate nucleus; EN, emboliform nucleus; FD, fascia dentata; FN, facial nucleus; GN, gracile nucleus; Hc, hippocampal formation; IO, inferior olivary nucleus; IZ, intermediate zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, marginal zone; PHG, parahippocampal gyrus; SB, subiculum; SP, subplate; STN, spinal trigeminal nucleus; SVZ, subventricular zone; Th, thalamus; VZ, ventricular zone.

Figure 2.

Expression of PCDH11X/Y in the fetal brain. Cerebral cortex (A, 14 PCW female; B, 16 PCW male; C, 19 PCW male; D, 12 PCW male), hippocampal formation (E, 18 PCW female), cerebellum and pons (F, 18 PCW female), and medulla oblongata (G, 27 PCW male; H and I, 18 PCW female). Scale bars: (A, FI): 3000 μm; (B, C): 5000 μm; (D) 100 μm; (E) 1000 μm. AN, abducens nucleus; ArN, arcuate nucleus; Cau, caudate; CCtx, cerebellar cortex; CN, cuneate nucleus; CP, cortical plate; DN, dentate nucleus; EN, emboliform nucleus; FD, fascia dentata; FN, facial nucleus; GN, gracile nucleus; Hc, hippocampal formation; IO, inferior olivary nucleus; IZ, intermediate zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, marginal zone; PHG, parahippocampal gyrus; SB, subiculum; SP, subplate; STN, spinal trigeminal nucleus; SVZ, subventricular zone; Th, thalamus; VZ, ventricular zone.

Expression of PCDH11X/Y in the Adult Human Brain

All PCDH11X/Y antibodies produced a pattern of immunoreactivity in male and female brains that was indistinguishable from each other, and reflected the immunoreactivity observed in the fetal brains. Strong PCDH11X/Y immunoreactivity was detected in neurons of layers II–VI in the frontal cortex (Fig. 3A), cingulate gyrus, occipital pole, and temporal cortex (Fig. 3B), with pyramidal cells prominently labeled (Figs 1GI and 3C). As in the fetal brains, strong immunoreactivity was observed in the Purkinje cells (Fig. 3F) and dentate nucleus. Moderate immunoreactivity was seen in the hippocampal formation (Fig. 3G), amygdala, the inferior olivary nucleus (Fig. 3I), and the caudate and putamen (Fig. 3D). Weak immunoreactivity was detected in the thalamus (Fig. 3H), the dorsal raphe nucleus (Fig. 3H), the hypoglossal nucleus (Fig. 3I), the nucleus of the solitary tract (Fig. 3I), and the spinal cord. PCDH11X/Y immunoreactivity was co-localized with the calcium-binding proteins calretinin (Fig. 1J), calbindin (Fig. 1K), and parvalbumin. Results are summarized in Table 2.

Figure 3.

Expression of PCDH11X/Y in the adult brain. Frontal cortex (A, male), superior temporal gyrus (B, female) with prominent expression in pyramidal neurons (C, female), basal ganglia (D, male), thalamus (E, female), cerebellar cortex (F, male), hippocampal formation (G, female), midbrain (H, female), and medulla oblongata (I, male). Scale bars: (A) 200 μm; (B) 100 μm; (C) 50 μm; (D, E GI) 5000 μm; (F) 25 μm. CA1-4, areas of Ammon's horn; Cau, caudate; DR, dorsal raphe; FD, fascia dentata; GL, granular layer; HN, hypoglossal nucleus; I–VI, cortical layers I–VI; IC, internal capsule; IO, inferior olivary nucleus; LD, lateral dorsal nucleus; MD, mediodorsal nucleus; ML, molecular layer; PL, Purkinje cell layer; Put, putamen; Red, red nucleus; SB, subiculum; SN, substantia nigra; WM, white matter.

Figure 3.

Expression of PCDH11X/Y in the adult brain. Frontal cortex (A, male), superior temporal gyrus (B, female) with prominent expression in pyramidal neurons (C, female), basal ganglia (D, male), thalamus (E, female), cerebellar cortex (F, male), hippocampal formation (G, female), midbrain (H, female), and medulla oblongata (I, male). Scale bars: (A) 200 μm; (B) 100 μm; (C) 50 μm; (D, E GI) 5000 μm; (F) 25 μm. CA1-4, areas of Ammon's horn; Cau, caudate; DR, dorsal raphe; FD, fascia dentata; GL, granular layer; HN, hypoglossal nucleus; I–VI, cortical layers I–VI; IC, internal capsule; IO, inferior olivary nucleus; LD, lateral dorsal nucleus; MD, mediodorsal nucleus; ML, molecular layer; PL, Purkinje cell layer; Put, putamen; Red, red nucleus; SB, subiculum; SN, substantia nigra; WM, white matter.

Discussion

The first immunohistochemical investigation of the expression of PCDH11X/Y protein in the human brain has demonstrated PCDH11X/Y immunoreactivity in the cytoplasm of neurons of the developing and adult cerebral cortex in both sexes and on both sides of the brain.

In the fetal brains, immunoreactivity was prominent in the medial and lateral ganglionic eminences (Fig. 2B) and the developing neocortex (Figs 1AF and 2AD), resembling the pattern of PCDH11X/Y expression observed in situ with a common PCDH11X/Y riboprobe at 16–20 weeks gestation by Dibbens et al. (2008). These authors also reported that PCDH11X/Y was expressed in the embryonic caudate nucleus exclusively in females; they detected no expression on northern blots of adult caudate from either sex. In our study, PCDH11X/Y immunoreactivity was detected in both sexes at all ages in which the caudate was present, including an 16-postconceptional weeks (PCW) male, and adults of both sexes. Furthermore, both PCDH11X and PCDH11Y transcripts have been detected in the adult caudate by RT-PCR and confirmed by restriction digests at sex-specific sites (Blanco et al. 2000; Blanco-Arias et al. 2004). This disparity may reflect mismatches between the presence of mRNA and the encoded protein that have been observed in brain (Tropea et al. 2001) and muscle (Andersen and Schiaffino 1997). Other areas of strong immunoreactivity in the fetal brains were the inferior olive (Fig. 2G and H) and the deep cerebellar nuclei (Fig. 2F). The strong immunoreactivity observed in the uppermost layers of the cortical plate and the ventricular zone at 12 PCW (e.g. Fig. 2D) may suggest PCDH11X/Y expression in recently migrated pyramidal neurons and their precursors, respectively. The co-localization of PCDH11X/Y and DCX (Fig. 1E), a microtubule-associated protein that is a marker of migrating neurons (Francis et al. 1999; Gleeson et al. 1999), in the subplate and cortical plate together with the strong immunoreactivity of pyramidal neurons in the adult cortex (Fig. 3B,C) supports this supposition. As the fetal cortex continues to develop, expression of PCDH11X/Y is also seen in the subplate, albeit less strongly than in the cortical plate and ventricular zone (e.g. Figs 1AF and 2B,C). NPY is principally confined to neurons residing in the subplate (Delalle et al. 1997; Bayatti et al. 2008), and double labeling with NPY suggests that these resident subplate neurons also express PCDH11X/Y (Fig. 1F).

In the adult brains, expression was detected in all cortical areas in layers II–VI (Fig. 3A) with pyramidal neurons prominently labeled (Figs 1GI and 2B,C). PCDH11X/Y expression was not confined to any subtype of interneurons as identified by the calcium-binding proteins, calretinin (Fig. 1J), calbindin (Fig. 1K), and parvalbumin.

We did not observe any asymmetric expression of PCDH11X/Y in the cerebral cortex nor was the expression in the superior temporal gyrus remarkable. The Purkinje cells (Fig. 3F) of the cerebellar cortex were intensely immunoreactive, as was the dentate nucleus. Immunoreactivity was also detected in the amygdala, hippocampal formation (Fig. 3G), caudate (Fig. 3D), and thalamus (Fig. 3H), coinciding with the prior RT-PCR data (Yoshida and Sugano 1999; Blanco et al. 2000; Blanco-Arias et al. 2004).

The pattern of expression in the developing human cortex was similar to that reported in the ferret (Krishna-K et al. 2009) and the rat (Kim et al. 2007), and our findings of expression in the adult hippocampal formation and amygdala are similar to observations made in the rat (Kim et al. 2010) and mouse (Hertel et al. 2008). Reports of Pcdh11 expression in the cortex of adult experimental animals are less consistent: Ranging from complete absence in rat (Kim et al. 2007), a subpopulation of neurons in layers IV–VI in the mouse somatosensory cortex (Krishna-K et al. 2011), to layers II–VI of the mouse motor cortex (Hertel and Redies 2011), and layers II–VI of the ferret visual cortex (Krishna-K et al. 2009). Interneuron expression was not specifically addressed by these studies; however, work on γ-Pcdhs demonstrates widespread interneuronal expression in many structures (Wang et al. 2002; Phillips et al. 2003; Lefebvre et al. 2008).

The prominent PCDH11X/Y expression we observed throughout the cerebral cortex may be a consequence of the putative gene dosage doubling at 6 MYA and/or the addition of the human-specific cysteines in the ectodomain. Further investigation of the human specificity of this gene pair may be directed at the predecessor cells (Bystron et al. 2006), the first neurons to populate the human cerebral cortex at Carnegie stage 12.

The antibodies used were broad in their range of specificity (Procad1a and anti-PCDH11X/Y: Common to all isoforms; Ex6: Most short forms; X11: Most long forms) and the location of individual PCDH11X/Y isoforms may yet reveal a more distinct expression pattern. Our repeated attempts to raise a PCDH11Y-specific antibody (i.e. Ex6) were unsuccessful. Despite careful screening and selection of clones that only reacted with male brains, antibodies were found to be also reactive with female brains. Ex6 immunoreactivity is ameliorated by incubation with the recombinant PCDH11Ya cytoplasmic protein suggesting specificity to a Y motif, but the high similarity between PCDH11Ya and PCDH11Xb within the cytodomain (and indeed the entire protein) makes it difficult to design antibodies and nucleotide probes that differentiate the PCDH11X and PCDH11Y. This is also a problem for longer isoforms, for example, a report of upregulation of PCDH11Xc in males using microarrays (Galfalvy et al. 2003) is dubious given that the probesets in question are 100% identical to PCDH11Yc (Lopes et al. 2006; Weickert et al. 2009) and, in another report, 3 PCDH11Y isoform-specific primer pairs produced product from female tissues (Ahn et al. 2010). To avoid such cross-reactivity, studies have isolated small fragments that exploit the few PCDH11X/Y sequence differences (Giouzeli et al. 2004) or used common riboprobes (Dibbens et al. 2008) or pan PCDH11X/Y antibodies (Chen et al. 2002) to examine PCDH11X/Y as a whole. Cyclophosphamide immunosuppression (Ou et al. 1991; Sleister and Rao 2001) has enabled the production of antibodies against highly similar neuronal antigens and should be considered when raising antibodies against PCDH11X/Y in the future.

In summary, PCDH11X/Y is widely expressed throughout both the developing and adult human brain, with strong expression observed in the cerebral cortex, ganglionic eminence, Purjkinje cells and dentate nucleus of the cerebellum, and the inferior olivary nucleus. It is interesting to note that many of the latter structures use γ-aminobutyric acid as their transmitter; however, our investigation did not find restricted PCDH11X/Y expression in a limited subset of interneurons and further work is required. The lack of an asymmetric distribution and the widespread expression in non cortical areas observed in the present study does not support PCDH11X/Y's role in human-specific faculties, but it is worth noting that the antibodies were broad in their specificity and individual isoforms may yet be found in a more restricted pattern in cortical areas related to language. In addition, Pcdh11X has been present in the mammalian radiation for some time and any functions attributed to the human-specific changes to PCDH11X and the entirely new PCDH11Y may build upon existing neuronal systems. These data confirm earlier reports using RT-PCR and in situ hybridization and identify PCDH11X/Y expression in areas that were previously untested. Methods for studying expression that are specific for the individual isoforms of PCDH11X and, more importantly, PCDH11Y together with the determination of the inactivation status and the functional significance of the PCDH11X ectodomain changes will be necessary to understand the role of the gene pair in disease and the evolution of the human brain.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

This work was supported by the UK charity SANE and the TJ Crow Psychosis Trust. Funding to pay the Open Access Publication charges for this article was provided by the TJ Crow Psychosis Trust.

Notes

We gratefully acknowledge Waney Squier, for providing the fetal tissue used in this study, Mary Walker, for providing technical assistance, Kirsty Hewitson and Chris Schofield, for expressing recombinant proteins, Mark Cranfield and Nigel Groome, for assistance in raising the monoclonal antibodies, and Nic Williams and Margaret Esiri, for helpful discussions during the early stages of the project. Conflict of Interest: None declared.

References

Ahn
K
Huh
J-W
Kim
D-S
Ha
H-S
Kim
Y-J
Lee
J-R
Kim
H-S
Quantitative analysis of alternative transcripts of human PCDH11X/Y genes
Am J Med Genet B
 , 
2010
, vol. 
153B
 (pg. 
736
-
744
)
Andersen
JL
Schiaffino
S
Mismatch between myosin heavy chain mRNA and protein distribution in human skeletal muscle fibers
Am J Physiol
 , 
1997
, vol. 
272
 (pg. 
C1881
-
1889
)
Bayatti
N
Moss
JA
Sun
L
Ambrose
P
Ward
JFH
Lindsay
S
Clowry
GJ
A molecular neuroanatomical study of the developing human neocortex from 8 to 17 postconceptional weeks revealing the early differentiation of the subplate and subventricular zone
Cereb Cortex
 , 
2008
, vol. 
18
 (pg. 
1536
-
1548
)
Beecham
GW
Naj
AC
Gilbert
JR
Haines
JL
Buxbaum
JD
Pericak-Vance
MA
PCDH11X variation is not associated with late-onset Alzheimer disease susceptibility
Psychiatr Genet
 , 
2010
, vol. 
20
 (pg. 
321
-
324
)
Biswas
S
Emond
MR
Jontes
JD
Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation
J Cell Biol
 , 
2010
, vol. 
191
 (pg. 
1029
-
1041
)
Blanco
P
Sargent
CA
Boucher
CA
Mitchell
M
Affara
NA
Conservation of PCDHX in mammals; expression of human X/Y genes predominantly in brain
Mamm Genome
 , 
2000
, vol. 
11
 (pg. 
906
-
914
)
Blanco-Arias
P
Sargent
CA
Affara
NA
Protocadherin X (PCDHX) and Y (PCDHY) genes; multiple mRNA isoforms encoding variant signal peptides and cytoplasmic domains
Mamm Genome
 , 
2004
, vol. 
15
 (pg. 
41
-
52
)
Bystron
I
Rakic
P
Molnar
Z
Blakemore
C
The first neurons of the human cerebral cortex
Nat Neurosci
 , 
2006
, vol. 
9
 (pg. 
880
-
886
)
Carrasquillo
MM
Zou
F
Pankratz
VS
Wilcox
SL
Ma
L
Walker
LP
Younkin
SG
Younkin
CS
Younkin
LH
Bisceglio
GD
, et al.  . 
Genetic variation in PCDH11X is associated with susceptibility to late-onset Alzheimer's disease
Nat Genet
 , 
2009
, vol. 
41
 (pg. 
192
-
198
)
Chance
SA
Crow
TJ
Distinctively human: cerebral lateralisation and language in Homo sapiens
J Anthropol Sci
 , 
2007
, vol. 
85
 (pg. 
83
-
100
)
Chen
M
Vacherot
F
de la Taille
A
Gil-Diez-de-Medina
S
Shen
R
Friedman
RA
Burchardt
M
Chopin
DK
Buttyan
R
The emergence of protocadherin-PC expression during the acquisition of apoptosis-resistance by prostate cancer cells
Oncogene
 , 
2002
, vol. 
21
 (pg. 
7861
-
7871
)
Chen
X
Molino
C
Liu
L
Gumbiner
BM
Structural elements necessary for oligomerization, trafficking, and cell sorting function of paraxial protocadherin
J Biol Chem
 , 
2007
, vol. 
282
 (pg. 
32128
-
32137
)
Crow
TJ
The ‘big bang’ theory of the origin of psychosis and the faculty of language
Schizophr Res
 , 
2008
, vol. 
102
 (pg. 
31
-
52
)
Crow
TJ
Handedness, language lateralisation and anatomical asymmetry: relevance of protocadherin XY to hominid speciation and the aetiology of psychosis: point of view
Br J Psychiatry
 , 
2002
, vol. 
181
 (pg. 
295
-
297
)
Delalle
I
Evers
P
Kostović
I
Uylings
HBM
Laminar distribution of neuropeptide Y-immunoreactive neurons in human prefrontal cortex during development
J Comp Neurol
 , 
1997
, vol. 
379
 (pg. 
515
-
522
)
Dibbens
LM
Tarpey
PS
Hynes
K
Bayly
MA
Scheffer
IE
Smith
R
Bomar
J
Sutton
E
Vandeleur
L
Shoubridge
C
, et al.  . 
X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment
Nat Genet
 , 
2008
, vol. 
40
 (pg. 
776
-
781
)
Durand
CM
Kappeler
C
Betancur
C
Delorme
R
Quach
H
Goubran-Botros
H
Melke
J
Nygren
G
Chabane
N
Bellivier
F
, et al.  . 
Expression and genetic variability of PCDH11Y, a gene specific to Homo sapiens and candidate for susceptibility to psychiatric disorders
Am J Med Genet B
 , 
2005
, vol. 
141B
 (pg. 
67
-
70
)
Emond
MR
Biswas
S
Jontes
JD
Protocadherin-19 is essential for early steps in brain morphogenesis
Dev Biol
 , 
2009
, vol. 
334
 (pg. 
72
-
83
)
Evers
P
Uylings
HBM
An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative
J Neurosci Methods
 , 
1997
, vol. 
72
 (pg. 
197
-
207
)
Francis
F
Koulakoff
A
Boucher
D
Chafey
P
Schaar
B
Vinet
M-C
Friocourt
G
McDonnell
N
Reiner
O
Kahn
A
, et al.  . 
Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons
Neuron
 , 
1999
, vol. 
23
 (pg. 
247
-
256
)
Frank
M
Kemler
R
Protocadherins
Curr Opin Cell Biol
 , 
2002
, vol. 
14
 (pg. 
557
-
562
)
Galfalvy
H
Erraji-Benchekroun
L
Smyrniotopoulos
P
Pavlidis
P
Ellis
S
Mann
JJ
Sibille
E
Arango
V
Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction
BMC Bioinformatics
 , 
2003
, vol. 
4
 pg. 
37
 
Giouzeli
M
Williams
NA
Lonie
LJ
DeLisi
LE
Crow
TJ
ProtocadherinX/Y, a candidate gene-pair for schizophrenia and schizoaffective disorder: a DHPLC investigation of genomic sequence
Am J Med Genet B
 , 
2004
, vol. 
129B
 (pg. 
1
-
9
)
Gleeson
JG
Lin
PT
Flanagan
LA
Walsh
CA
Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons
Neuron
 , 
1999
, vol. 
23
 (pg. 
257
-
271
)
Gumbiner
BM
Regulation of cadherin-mediated adhesion in morphogenesis
Nat Rev Mol Cell Biol
 , 
2005
, vol. 
6
 (pg. 
622
-
634
)
Harlow
E
Lane
D
Monoclonal antibodies
Antibodies: a laboratory manual
 , 
1988
New York
Cold Spring Harbour Laboratory Press
pg. 
211
 
Hauser
MD
Chomsky
N
Fitch
WT
The faculty of language: what is it, who has it, and how did it evolve?
Science
 , 
2002
, vol. 
298
 (pg. 
1569
-
1579
)
Hertel
N
Krishna
K
Nuernberger
M
Redies
C
A cadherin-based code for the divisions of the mouse basal ganglia
J Comp Neurol
 , 
2008
, vol. 
508
 (pg. 
511
-
528
)
Hertel
N
Redies
C
Absence of layer-specific cadherin expression profiles in the neocortex of the reeler mutant mouse
Cereb Cortex
 , 
2011
, vol. 
21
 (pg. 
1105
-
1117
)
Hoffman
GE
Le
WW
Sita
LV
The importance of titrating antibodies for immunocytochemical methods
Curr Protoc Neurosci
 , 
2008
, vol. 
45
 (pg. 
2.12.11
-
26
)
Hulpiau
P
van Roy
F
Molecular evolution of the cadherin superfamily
Int J Biochem Cell Biol
 , 
2009
, vol. 
41
 (pg. 
349
-
369
)
Isles
AR
Wilkinson
LS
Epigenetics: what is it and why is it important to mental disease?
Br Med Bull
 , 
2008
, vol. 
85
 (pg. 
35
-
45
)
Itti
E
Gaw Gonzalo
IT
Boone
KB
Geschwind
DH
Berman
N
Pawlikowska-Haddal
A
Itti
L
Mishkin
FS
Swerdloff
RS
Functional neuroimaging provides evidence of anomalous cerebral laterality in adults with Klinefelter's syndrome
Ann Neurol
 , 
2003
, vol. 
54
 (pg. 
669
-
673
)
Itti
E
Gaw Gonzalo
IT
Pawlikowska-Haddal
A
Boone
KB
Mlikotic
A
Itti
L
Mishkin
FS
Swerdloff
RS
The structural brain correlates of cognitive deficits in adults with Klinefelter's syndrome
J Clin Endocrinol Metab
 , 
2006
, vol. 
91
 (pg. 
1423
-
1427
)
Kalmady
SV
Venkatasubramanian
G
Evidence for positive selection on protocadherin Y gene in Homo sapiens: implications for schizophrenia
Schizophr Res
 , 
2009
, vol. 
108
 (pg. 
299
-
300
)
Kesler
SR
Haberecht
MF
Menon
V
Warsofsky
IS
Dyer-Friedman
J
Neely
EK
Reiss
AL
Functional neuroanatomy of spatial orientation processing in turner syndrome
Cereb Cortex
 , 
2004
, vol. 
14
 (pg. 
174
-
180
)
Kim
SY
Chung
HS
Sun
W
Kim
H
Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain
Neuroscience
 , 
2007
, vol. 
147
 (pg. 
996
-
1021
)
Kim
SY
Mo
JW
Han
S
Choi
SY
Han
SB
Moon
BH
Rhyu
IJ
Sun
W
Kim
H
The expression of non-clustered protocadherins in adult rat hippocampal formation and the connecting brain regions
Neuroscience
 , 
2010
, vol. 
170
 (pg. 
189
-
199
)
Kopsida
E
Stergiakouli
E
Lynn
PM
Wilkinson
LS
Davies
W
The role of the Y chromosome in brain function
Open Neuroendocr J
 , 
2009
, vol. 
2
 (pg. 
20
-
30
)
Krishna-K
Nuernberger
M
Weth
F
Redies
C
Layer-specific expression of multiple cadherins in the developing visual cortex (V1) of the ferret
Cereb Cortex
 , 
2009
, vol. 
19
 (pg. 
388
-
401
)
Krishna-K
K
Hertel
N
Redies
C
Cadherin expression in the somatosensory cortex: evidence for a combinatorial molecular code at the single-cell level
Neuroscience
 , 
2011
, vol. 
175
 (pg. 
37
-
48
)
Lefebvre
JL
Zhang
Y
Meister
M
Wang
X
Sanes
JR
γ-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina
Development
 , 
2008
, vol. 
135
 (pg. 
4141
-
4151
)
Lescai
F
Pirazzini
C
D'Agostino
G
Santoro
A
Ghidoni
R
Benussi
L
Galimberti
D
Federica
E
Marchegiani
F
Cardelli
M
, et al.  . 
Failure to replicate an association of rs5984894 SNP in the PCDH11X gene in a collection of 1,222 Alzheimer's disease affected patients
J Alzheimer's Dis
 , 
2010
, vol. 
21
 (pg. 
385
-
388
)
Lopes
A
Ross
N
Close
J
Dagnall
A
Amorim
A
Crow
T
Inactivation status of PCDH11X: sexual dimorphisms in gene expression levels in brain
Hum Genet
 , 
2006
, vol. 
119
 (pg. 
265
-
275
)
Lopes
AM
Calafell
F
Amorim
A
Microsatellite variation and evolutionary history of PCDHX/Y gene pair within the Xq21.3/Yp11.2 hominid-specific homology block
Mol Biol Evol
 , 
2004
, vol. 
21
 (pg. 
2092
-
2101
)
Miar
A
Álvarez
V
Corao
AI
Alonso
B
Díaz
M
Menéndez
M
Martínez
C
Calatayud
M
Morís
G
Coto
E
Lack of association between protocadherin 11-X/Y (PCDH11X and PCDH11Y) polymorphisms and late onset Alzheimer's disease
Brain Res
 , 
2011
, vol. 
1383
 (pg. 
252
-
256
)
Morishita
H
Yagi
T
Protocadherin family: diversity, structure, and function
Curr Opin Cell Biol
 , 
2007
, vol. 
19
 (pg. 
584
-
592
)
Murphy
DGM
Mentis
MJ
Pietrini
P
Grady
C
Daly
E
Haxby
JV
De La Granja
M
Allen
G
Largay
K
White
BJ
A PET study of Turner's syndrome: effects of sex steroids and the X chromosome on brain
Biol Psychiatry
 , 
1997
, vol. 
41
 (pg. 
285
-
298
)
Nollet
F
Kools
P
van Roy
F
Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members
J Mol Biol
 , 
2000
, vol. 
299
 (pg. 
551
-
572
)
Otter
M
Schrander-Stumpel
CTRM
Curfs
LMG
Triple X syndrome: a review of the literature
Eur J Hum Genet
 , 
2010
, vol. 
18
 (pg. 
265
-
271
)
Ou
SK
McDonald
C
Patterson
PH
Comparison of two techniques for targeting the production of monoclonal antibodies against particular antigens
J Immunol Methods
 , 
1991
, vol. 
145
 (pg. 
111
-
118
)
Phillips
GR
Tanaka
H
Frank
M
Elste
A
Fidler
L
Benson
DL
Colman
DR
γ-Protocadherins are targeted to subsets of synapses and intracellular organelles in neurons
J Neurosci
 , 
2003
, vol. 
23
 (pg. 
5096
-
5104
)
Priddle
TH
Crow
TJ
The protocadherin 11X/Y gene pair as a putative determinant of cerebral dominance in Homo sapiens
Future Neurol
 , 
2009
, vol. 
4
 (pg. 
509
-
518
)
Priddle
TH
Lee
WH
Crow
TJ
Yoshida
K
The Protocadherin 11X/Y gene pair and the evolution of the hominin brain
Molecular and functional diversities of cadherin and protocadherin
 , 
2010
Trivandrum (India)
Research Signpost
(pg. 
313
-
344
)
Rae
C
Joy
P
Harasty
J
Kemp
A
Kuan
S
Christodoulou
J
Cowell
CT
Coltheart
M
Enlarged temporal lobes in turner syndrome: an X-chromosome effect?
Cereb Cortex
 , 
2004
, vol. 
14
 (pg. 
156
-
164
)
Rashid
D
Newell
K
Shama
L
Bradley
R
A requirement for NF-protocadherin and TAF1/Set in cell adhesion and neural tube formation
Dev Biol
 , 
2006
, vol. 
291
 (pg. 
170
-
181
)
Redies
C
Vanhalst
K
van Roy
F
δ-Protocadherins: unique structures and functions
Cell Mol Life Sci
 , 
2005
, vol. 
62
 (pg. 
2840
-
2852
)
Rezaie
R
Daly
EM
Cutter
WJ
Murphy
DGM
Robertson
DMW
DeLisi
LE
Mackay
CE
Barrick
TR
Crow
TJ
Roberts
N
The influence of sex chromosome aneuploidy on brain asymmetry
Am J Med Genet B
 , 
2008
, vol. 
150B
 (pg. 
74
-
85
)
Ross
NLJ
Wadekar
R
Lopes
A
Dagnall
A
Close
J
DeLisi
LE
Crow
TJ
Methylation of two Homo sapiens-specific X-Y homologous genes in Klinefelter's syndrome (XXY)
Am J Med Genet B
 , 
2006
, vol. 
141B
 (pg. 
544
-
548
)
Schnell
SA
Staines
WA
Wessendorf
MW
Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue
J Histochem Cytochem
 , 
1999
, vol. 
47
 (pg. 
719
-
730
)
Schreiner
D
Weiner
JA
Combinatorial homophilic interaction between γ-protocadherin multimers greatly expands the molecular diversity of cell adhesion
Proc Natl Acad Sci USA
 , 
2010
, vol. 
107
 (pg. 
14893
-
14898
)
Sleister
HM
Rao
AG
Strategies to generate antibodies capable of distinguishing between proteins with >90% amino acid identity
J Immunol Methods
 , 
2001
, vol. 
252
 (pg. 
121
-
129
)
Speevak
MD
Farrell
SA
Non-syndromic language delay in a child with disruption in the protocadherin11X/Y gene pair
Am J Med Genet B
 , 
2011
, vol. 
156
 (pg. 
484
-
489
)
Trombetta
B
Cruciani
F
Underhill
PA
Sellitto
D
Scozzari
R
Footprints of X-to-Y gene conversion in recent human evolution
Mol Biol Evol
 , 
2010
, vol. 
27
 (pg. 
714
-
725
)
Tropea
D
Capsoni
S
Tongiorgi
E
Giannotta
S
Cattaneo
A
Domenici
L
Mismatch between BDNF mRNA and protein expression in the developing visual cortex: the role of visual experience
Eur J Neurosci
 , 
2001
, vol. 
13
 (pg. 
709
-
721
)
Turner
JMA
Meiotic sex chromosome inactivation
Development
 , 
2007
, vol. 
134
 (pg. 
1823
-
1831
)
Vanhalst
K
Kools
P
Staes
K
van Roy
F
Redies
C
δ-Protocadherins: a gene family expressed differentially in the mouse brain
Cell Mol Life Sci
 , 
2005
, vol. 
62
 (pg. 
1247
-
1259
)
Visootsak
J
Graham
J
Klinefelter syndrome and other sex chromosomal aneuploidies
Orphanet J Rare Dis
 , 
2006
, vol. 
1
 pg. 
42
 
Wang
X
Weiner
JA
Levi
S
Craig
AM
Bradley
A
Sanes
JR
Gamma protocadherins are required for survival of spinal interneurons
Neuron
 , 
2002
, vol. 
36
 (pg. 
843
-
854
)
Weickert
CS
Elashoff
M
Richards
AB
Sinclair
D
Bahn
S
Paabo
S
Khaitovich
P
Webster
MJ
Transcriptome analysis of male-female differences in prefrontal cortical development
Mol Psychiatry
 , 
2009
, vol. 
14
 (pg. 
558
-
561
)
Weiner
JA
Wang
X
Tapia
JC
Sanes
JR
Gamma protocadherins are required for synaptic development in the spinal cord
Proc Natl Acad Sci USA
 , 
2005
, vol. 
102
 (pg. 
8
-
14
)
Whibley
AC
Plagnol
V
Tarpey
PS
Abidi
F
Fullston
T
Choma
MK
Boucher
CA
Shepherd
L
Willatt
L
Parkin
G
, et al.  . 
Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability
Am J Hum Genet
 , 
2010
, vol. 
87
 (pg. 
173
-
188
)
Williams
NA
Close
JP
Giouzeli
M
Crow
TJ
Accelerated evolution of protocadherin11X/Y: a candidate gene-pair for cerebral asymmetry and language
Am J Med Genet B
 , 
2006
, vol. 
141B
 (pg. 
623
-
633
)
Wilson
N
Ross
L
Close
J
Mott
R
Crow
T
Volpi
E
Replication profile of PCDH11X and PCDH11Y, a gene pair located in the non-pseudoautosomal homologous region Xq21.3/Yp11.2
Chromosome Res
 , 
2007
, vol. 
15
 (pg. 
485
-
498
)
Wilson
ND
Ross
LJN
Crow
TJ
Volpi
EV
PCDH11 is X/Y homologous in Homo sapiens but not in Gorilla gorilla and Pan troglodytes
Cytogenet Genome Res
 , 
2006
, vol. 
114
 pg. 
137
 
Wu
Q
Maniatis
T
A striking organization of a large family of human neural cadherin-like cell adhesion genes
Cell
 , 
1999
, vol. 
97
 (pg. 
779
-
790
)
Wu
Z-C
Yu
J-T
Wang
N-D
Yu
N-N
Zhang
Q
Chen
W
Zhang
W
Zhu
Q-X
Tan
L
Lack of association between PCDH11X genetic variation and late-onset Alzheimer's disease in a Han Chinese population
Brain Res
 , 
2010
, vol. 
1357
 (pg. 
152
-
156
)
Yang
X
Chen
M-W
Terry
S
Vacherot
F
Chopin
DK
Bemis
DL
Kitajewski
J
Benson
MC
Guo
Y
Buttyan
R
A human- and male-specific protocadherin that acts through the wnt signaling pathway to induce neuroendocrine transdifferentiation of prostate cancer cells
Cancer Res
 , 
2005
, vol. 
65
 (pg. 
5263
-
5271
)
Yoshida
K
Sugano
S
Identification of a novel protocadherin gene (PCDH11) on the human XY homology region in Xq21.3
Genomics
 , 
1999
, vol. 
62
 (pg. 
540
-
543
)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.