Trimethylation and Acetylation of ß-Catenin at Lysine 49 Represent Key Elements in ESC Pluripotency.

Wnt/ß-catenin signaling is required for embryonic stem cell (ESC) pluripotency by inducing mesodermal differentiation and inhibiting neuronal differentiation; however, how ß-catenin counter-regulates these differentiation pathways is unknown. Here, we show that lysine 49 (K49) of ß-catenin is trimethylated (ß-catMe3) by Ezh2 or acetylated (ß-catAc) by Cbp. Significantly, ß-catMe3 acts as a transcriptional co-repressor of the neuronal differentiation genes sox1 and sox3, whereas ß-catAc acts as a transcriptional co-activator of the key mesodermal differentiation gene t-brachyury (t-bra). Furthermore, ß-catMe3 and ß-catAc are alternatively enriched on repressed or activated genes, respectively, during ESC and adult stem cell differentiation into neuronal or mesodermal progenitor cell lineages. Importantly, expression of a ß-catenin K49A mutant results in major defects in ESC differentiation. We conclude that ß-catenin K49 trimethylation and acetylation are key elements in regulating ESC pluripotency and differentiation potential.

Editor's Highlight: Identification and Characterization of Teratogenic Chemicals Using Embryonic Stem Cells Isolated From a Wnt/ß-Catenin-Reporter Transgenic Mouse Line.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/ß-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a ß-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/ß-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/ß-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

A Bmp Reporter Transgene Mouse Embryonic Stem Cell Model as a Tool to Identify and Characterize Chemical Teratogens.

Embryonic stem cells (ESCs) were first isolated from mouse embryos more than 30 years ago. They have proven invaluable not only in generating genetically modified mice that allow for analysis of gene function in tissue development and homeostasis but also as models for genetic disease. In addition, ESCs in vitro are finding inroads in pharmaceutical and toxicological testing, including the identification of teratogenic compounds. Here, we describe the use of a bone morphogenetic protein (Bmp)-reporter ESC line, isolated from a well-characterized transgenic mouse line, as a new tool for the identification of chemical teratogens. The Bmp-mediated expression of the green fluorescent protein enabled the quantification of dose- and time-dependent effects of valproic acid as well as retinoic acid. Significant effects were detectable at concentrations that were comparable to the ones observed in the classical embryonic stem cell test, despite the fact that the reporter gene is expressed in distinct cell types, including endothelial and endodermal cells. Thus these cells provide a valuable new tool for the identification and characterization of relevant mechanisms of embryonic toxicity.

Beta-catenin is vital for the integrity of mouse embryonic stem cells.

ß-Catenin mediated Wnt-signaling is assumed to play a major function in embryonic stem cells in maintaining their stem cell character and the exit from this unique trait. The complexity of ß-catenin action and conflicting results on the role of ß-catenin in maintaining the pluripotent state have made it difficult to understand its precise cellular and molecular functions. To attempt this issue we have generated new genetically modified mouse embryonic stem cell lines allowing for the deletion of ß-catenin in a controlled manner by taking advantage of the Cre-ER-T2 system and analyzed the effects in a narrow time window shortly after ablation. By using this approach, rather then taking long term cultured ß-catenin null cell lines we demonstrate that ß-catenin is dispensable for the maintenance of pluripotency associated genes. In addition we observed that the removal of ß-catenin leads to a strong increase of cell death, the appearance of multiple clustered functional centrosomes most likely due to a mis-regulation of the polo-like-kinase 2 and furthermore, alterations in chromosome segregation. Our study demonstrates the importance of ß-catenin in maintaining correct cellular functions and helps to understand its role in embryonic stem cells.

Wnt3a-dependent and -independent protein interaction networks of chromatin-bound ß-catenin in mouse embryonic stem cells.

Canonical Wnt signaling is repeatedly used during development to control cell fate, and it is often implicated in human cancer. ß-catenin, the effector of Wnt signaling, has a dual function in the cell and is involved in both cell adhesion and transcription. Nuclear ß-catenin controls transcription through association with transcription factors of the TCF family and the recruitment of epigenetic modifiers. In this study, we used a strategy combining the genetic manipulation of mouse embryonic stem cells with affinity purification and quantitative mass spectroscopy utilizing stable isotope labeling with amino acids in cell culture to study the interactome of chromatin-bound ß-catenin with and without Wnt3a stimulation. We uncovered previously unknown interactions of ß-catenin with transcription factors and chromatin-modifying complexes. Our proof-of-principle experiments show that ß-catenin can recruit the H3K4me2/1 demethylase LSD1 to regulate the expression of the tumor suppressor Lefty1 in mouse embryonic stem cells. The mRNA levels of LSD1 and ß-catenin are inversely correlated with the levels of Lefty1 in pancreas and breast tumors, implying that this mechanism is common to mouse embryonic stem cells and cancer cells.

E-cadherin is required for the proper activation of the Lifr/Gp130 signaling pathway in mouse embryonic stem cells.

The leukemia inhibitory factor (Lif) signaling pathway is a crucial determinant for mouse embryonic stem (mES) cell self-renewal and pluripotency. One of the hallmarks of mES cells, their compact growth morphology, results from tight cell adhesion mediated through E-cadherin, ß-catenin (Ctnnb1) and α-catenin with the actin cytoskeleton. ß-catenin is also involved in canonical Wnt signaling, which has also been suggested to control mES cell stemness. Here, we analyze Ctnnb1(-/-) mES cells in which cell adhesion is preserved by an E-cadherin-α-catenin (Eα) fusion protein (Ctnnb1(-/-)Eα mES cells), and show that mimicking only the adhesive function of ß-catenin is necessary and sufficient to maintain the mES cell state, making ß-catenin/Wnt signaling obsolete in this process. Furthermore, we propose a role for E-cadherin in promoting the Lif signaling cascade, showing an association of E-cadherin with the Lifr-Gp130 receptor complex, which is most likely facilitated by the extracellular domain of E-cadherin. Without Eα, and thus without maintained cell adhesion, Ctnnb1(-/-) mES cells downregulate components of the Lif signaling pathway, such as Lifr, Gp130 and activated Stat3, as well as pluripotency-associated markers. From these observations, we hypothesize that the changes in gene expression accompanying the loss of pluripotency are a direct consequence of dysfunctional cell adhesion. Supporting this view, we find that the requirement for intact adhesion can be circumvented by the forced expression of constitutively active Stat3. In summary, we put forward a model in which mES cells can be propagated in culture in the absence of Ctnnb1, as long as E-cadherin-mediated cell adhesion is preserved.

Differential requirements for ß-catenin during mouse development.

Embryogenesis relies on the precise interplay of signaling cascades to activate tissue-specific differentiation programs. An important player in these morphogenetic processes is ß-catenin, which is a central component of adherens junctions and canonical Wnt signaling. Lack of ß-catenin is lethal before gastrulation, but mice heterozygous for ß-catenin (Ctnnb1) develop as wild type. Here, we confine ß-catenin amounts below the heterozygous expression level to study the functional consequences for development. We generate embryonic stem (ES) cells and embryos expressing ß-catenin only from the ubiquitously active ROSA26 promoter and thereby limit ß-catenin expression to ~12.5% (ROSA26(ß/+)) or ~25% (ROSA26(ß/ß)) of wild-type levels. ROSA26(ß/+) is sufficient to maintain ES cell morphology and pluripotent characteristics, but is insufficient to activate canonical target genes upon Wnt stimulation. This Wnt signaling deficiency is incompletely restored in ROSA26(ß/ß) ES cells. We conclude that even very low ß-catenin levels are able to sustain cell adhesion, but not Wnt signaling. During development, ROSA26(ß/ß) as well as ROSA26(ß/+) partially rescues the knockout phenotype, yet proper gastrulation is absent. These embryos differentiate according to the neural default hypothesis, indicating that gastrulation depends on high ß-catenin levels. Strikingly, if ROSA26(ß/+) or ROSA26(ß/ß) is first activated after gastrulation, subsequent development correlates with the dosage of ß-catenin. Moreover, molecular evidence indicates that the amount of ß-catenin controls the induction of specific Wnt target genes. In conclusion, by restricting its expression we determine the level of ß-catenin required for adhesion or pluripotency and during different morphogenetic events.

Wnt/ß-catenin signaling regulates telomerase in stem cells and cancer cells.

Telomerase activity controls telomere length and plays a pivotal role in stem cells, aging, and cancer. Here, we report a molecular link between Wnt/ß-catenin signaling and the expression of the telomerase subunit Tert. ß-Catenin-deficient mouse embryonic stem (ES) cells have short telomeres; conversely, ES cell expressing an activated form of ß-catenin (ß-cat(ΔEx3/+)) have long telomeres. We show that ß-catenin regulates Tert expression through the interaction with Klf4, a core component of the pluripotency transcriptional network. ß-Catenin binds to the Tert promoter in a mouse intestinal tumor model and in human carcinoma cells. We uncover a previously unknown link between the stem cell and oncogenic potential whereby ß-catenin regulates Tert expression, and thereby telomere length, which could be critical in human regenerative therapy and cancer.

Lineage specification of parietal epithelial cells requires ß-catenin/Wnt signaling.

ß-Catenin/Wnt signaling is essential during early inductive stages of kidney development, but its role during postinductive stages of nephron development and maturation is not well understood. In this study, we used Pax8Cre mice to target ß-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional ß-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of tubules was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated podocytes replaced parietal epithelial cells in Bowman's capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional ß-catenin knockout mice revealed that these "parietal podocytes" derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that ß-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

Replacement of E-cadherin by N-cadherin in the mammary gland leads to fibrocystic changes and tumor formation.

INTRODUCTION: E-cadherin (E-cad; cadherin 1) and N-cadherin (N-cad; cadherin 2) are the most prominent members of the cadherin family of cell adhesion molecules. Although they share many structural and functional features, they are expressed in an almost mutually exclusive manner in vivo. METHODS: To explore functional differences between the two cadherins in vivo, we recently generated a knock-in line in which N-cad is expressed from the E-cad locus. In combination with a conditional gene inactivation approach, we expressed N-cad in the absence of E-cad (referred to as Ncadk.i.) in alveolar epithelial cells of the mammary gland starting in late pregnancy. RESULTS: We found that the sole presence of N-cad induces constitutively active fibroblast growth factor (Fgf) signaling and a precocious involution resulting in massive apoptosis of alveolar cells. To block apoptosis, we conditionally deleted one allele of p53 in Ncadk.i. mice and observed a temporal rescue of alveolar morphology and function. However, an accumulation of fibrotic tissue and cysts with increasing age and lactation cycles was observed. This phenotype closely resembled fibrocystic mastopathy (FM), a common disorder in humans, which is thought to precede breast cancer. Concordantly, 55% of Ncadk.i. mice harboring a heterozygous p53 deletion developed malignant and invasive tumors. CONCLUSIONS: Our results demonstrate a possible role for N-cad in the formation of fibrosis and cysts in the mammary gland. Moreover, we show that these lesions precede the development of malignant tumors. Thus, we provide a new mouse model to investigate the molecular mechanisms of fibrocystic mastopathy and the transition from benign to malignant tumors.

Biological and mathematical modeling of melanocyte development.

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function ß-catenin mutants in the melanocyte lineage. We found that any alteration of ß-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through ß-catenin.

Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation.

Self-renewal of rodent embryonic stem cells is enhanced by partial inhibition of glycogen synthase kinase-3 (Gsk3; refs 1, 2). This effect has variously been attributed to stimulation of Wnt signalling by ß-catenin, stabilization of Myc protein and global de-inhibition of anabolic processes. Here we demonstrate that ß-catenin is not necessary for embryonic stem cell identity or expansion, but its absence eliminates the self-renewal response to Gsk3 inhibition. Responsiveness is fully restored by truncated ß-catenin lacking the carboxy-terminal transactivation domain. However, requirement for Gsk3 inhibition is dictated by expression of T-cell factor 3 (Tcf3) and mediated by direct interaction with ß-catenin. Tcf3 localizes to many pluripotency genes in embryonic stem cells. Our findings confirm that Tcf3 acts as a transcriptional repressor and reveal that ß-catenin directly abrogates Tcf3 function. We conclude that Gsk3 inhibition stabilizes the embryonic stem cell state primarily by reducing repressive influence on the core pluripotency network.

N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation.

We conditionally substituted E-cadherin (E-cad; cadherin 1) with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of beta-catenin (beta-cat; Ctnnb1) and enhanced expression of several beta-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. Our experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations.

Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism.

Early lineage segregation in mouse development results in two, either CDX2- or OCT4/NANOG-positive, cell populations. CDX2-positive cells form the trophectoderm (TE), OCT4/NANOG-positive cells the inner cell mass (ICM). In a second lineage decision ICM cells segregate into Epiblast (EPI) and primitive endoderm (PE). EPI and PE formation depend on the activity of the transcription factors Nanog and Gata4/6. A role for Nanog, a crucial pluripotency factor, in preventing PE differentiation has been proposed, as outgrowths of mutant ICMs result in PE, but not EPI derivatives. We established Nanog-mutant mouse lines and analyzed EPI and PE formation in vivo. Surprisingly, Gata4 expression in mutant ICM cells is absent or strongly decreased, thus loss of Nanog does not result in precocious endoderm differentiation. However, Nanog-deficient embryos retain the capacity to form PE in chimeric embryos and, in contrast to recent reports, in blastocyst outgrowths. Based on our findings we propose a non-cell autonomous requirement of Nanog for proper PE formation in addition to its essential role in EPI determination.

Beta-catenin-mediated signaling and cell adhesion in postgastrulation mouse embryos.

beta-Catenin plays two major roles during the development of multicellular organisms. It is the downstream effector of the canonical Wnt signaling cascade, which is involved in many developmental processes and in tumor formation. Additionally, it is linked to classic cadherins and is required for the correct assembly and function of adherens junctions. beta-Catenin loss of function mutants show early gastrulation lethality. To address the role of beta-catenin in postgastrulation stages and to overcome the early embryonic lethality, we performed conditional gene targeting, using Cdx1::Cre, a newly established mouse line. By this approach, beta-catenin was depleted in the entire posterior embryo after the gastrulation process at embryonic day 8.0, when the three germ layers were established. We observed defects in signaling and adhesion which are temporarily separated. At an early event, known targets of Wnt/beta-catenin are down-regulated in the paraxial mesoderm. Moreover, Fgf8 and Wnt3a, the key players of the segmentation process, are down-regulated in the neural ectoderm (NE). Wnt3a expression was rescued in mutant embryos by exogenous Fgf and inhibition of Fgf signaling in wild-type embryos resulted in Wnt3a down-regulation. Based on these results, we assume the existence of an autoregulatory feedback loop in the NE where Fgf8 regulates Wnt3a, which in turn, by means of beta-catenin, maintains Fgf8 expression. In later stages, the lack of beta-catenin caused a progressive posterior disintegration. We found that beta-catenin is required for the correct localization of N-cadherin at the membrane of neural ectodermal cells and that its absence causes a disintegration of the neural tube.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid-gastrulation.

Transgenic mice with a defined cell- or tissues-specific expression of Cre-recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre-expression from Cdx1 regulatory elements. The expression of Cre-recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre-mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development.

Neural progenitors of the postnatal and adult mouse forebrain retain the ability to self-replicate, form neurospheres, and undergo multipotent differentiation in vivo.

Somatic stem cells are reservoirs to replace lost cells or damaged tissue. Cells with neural stem cell (NSC) characteristics can be isolated from the postnatal mammalian brain into adulthood and expanded as neurospheres. We addressed the ability of these in vitro expanded putative NSCs to retain progenitor characteristics in vivo, in analogy to hematopoietic stem cells. When transplanted in utero, both postnatal and adult neural progenitors colonize host brains and contribute neurons and glia. In stark contrast to what has been reported when transplanted in postnatal hosts, epidermal growth factor-expanded cells also remain self-replicating and multipotent in vivo over many months and can be serially transplanted into multiple hosts. Surprisingly, embryonically transplanted NSCs remain in the neurogenic regions in adult hosts, where they express progenitor cell markers and continue to proliferate even after 6 months without tumor formation. These data indicate that spherogenic cells of the postnatal and adult mammalian brain retain their potential in vitro and in vivo throughout the life of the organism and beyond transplantation, which has important implications for cell replacement strategies.

Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system.

The formation of synapses is dependent on the expression of surface adhesion molecules that facilitate correct recognition, stabilization and function. The more than 60 clustered protocadherins (Pcdhalpha, Pcdhbeta and Pcdhgamma) identified in human and mouse have attracted considerable attention because of their clustered genomic organization and the potential role of alpha- and gamma-Pcdhs in allocating a neuronal surface code specifying synaptic connectivity. Here, we investigated whether beta-Pcdhs also contribute to these processes. By performing RT-PCR, we found a striking parallel onset of expression of many beta-Pcdhs around the onset of neurogenesis and wide expression in the central nervous system. We generated antibodies specific to Pcdhb16 and showed localization of Pcdhb16 protein in the adult mouse cerebellum, hippocampus and cerebral cortex. Analysing the mouse retina in detail revealed localization of Pcdhb16 to specific cell types and, importantly, subsets of synapses. We show that Pcdhb16 localizes predominantly to postsynaptic compartments and the comparison with Pcdhb22 implies differential localization and functions of individual beta-Pcdhs in the mammalian central nervous system. Moreover, we provide evidence for a role of beta-Pcdhs in the outer segments and connecting cilia of photoreceptors. Our data show for the first time that beta-Pcdhs also localize to specific neuronal subpopulations and synapses, providing support for the hypothesis that clustered Pcdhs are candidate genes for the specification of synaptic connectivity and neuronal networks.