Identification of a novel intermediate filament-linked N-cadherin/gamma-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells

Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both β- and γ-catenins are reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, γ-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ-catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach made possible, for the first time, the separation of N-cadherin/γ-catenin from N-cadherin/β-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/γ-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, γ-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while β-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/γ-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes.     

Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens

Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo.     

A novel type of adhering junction in an epithelioid tumorigenic rat cell culture line

Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with α- and β-catenin, vinculin and α-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1–3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, α- and β-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, “pan-cadherin” antibodies have reacted with a band at ～140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.     

Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (β-casein expression) was evaluated. Heterocellular interaction is critical for β-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (α-catenin, β-catenin and ZO−2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although β-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and β-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear β-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of β-catenin in GJ complexes.     

DHT deficiency perturbs the integrity of the rat seminiferous epithelium by disrupting tight and adherens junctions

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, β-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.     

Distribution of the cadherin-catenin complex in normal human thyroid epithelium and a thyroid carcinoma cell line

E-cadherin is the major cell-cell adhesion molecule expressed by epithelial cells. Cadherins form a complex with three cytoplasmic proteins, α-, β-, and γ-catenin, and the interaction between them is crucial for anchoring the actin cytoskeleton to the intercellular adherens junctions. The invasive behavior of cancer cells has been attributed to a dysfunction of these molecules. In this study, we examined the distribution of the cadherin-catenin complex in a Chinese human thyroid cancer cell line, CGTH W-2, compared with that in normal human thyroid epithelial cells. In the normal cells, using immunofluorescence staining, E-cadherin and α-, β-, and γ-catenin were found to be localized at the intercellular junction and appeared as 135, 102, 90, and 80 kD proteins on Western blots. In CGTH W-2 cells, no E-cadherin and γ-catenin immunoreactivity was detected by immunofluorescence or Western blotting; α- and β-catenin were detected as 102 and 90 kD proteins on blots but gave a diffuse cytoplasmic immunofluorescence staining pattern in most cells, while β-catenin was also distributed throughout the cytoplasm in most cells but was found at the cell junction in some, where it colocalized with α-actinin. The present data indicate that the loss of cell adhesiveness in these cancer cells may be due to incomplete assembly of the cadherin-catenin complex at the cell junction. However, this defect did not affect the linkage of actin bundles to vinculin-enriched intercellular junctions. J. Cell. Biochem. 70:330–337, 1998. © 1998 Wiley-Liss, Inc.     

Linkage of N-cadherin to multiple cytoskeletal elements revealed by a proteomic approach in hippocampal neurons

The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of β- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na(+)/K(+)-ATPase bridged with spectrins. A close relative of β-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated β-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation

Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD2⁻ NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target.     

Adherens and tight junctions: Structure, function and connections to the actin cytoskeleton

Adherens junctions and Tight junctions comprise two modes of cell-cell adhesion that provide different functions. Both junctional complexes are proposed to associate with the actin cytoskeleton, and formation and maturation of cell-cell contacts involves reorganization of the actin cytoskeleton. Adherens junctions initiate cell-cell contacts, and mediate the maturation and maintenance of the contact. Adherens junctions consist of the transmembrane protein E-cadherin, and intracellular components, p120-catenin, β-catenin and α-catenin. Tight junctions regulate the paracellular pathway for the movement of ions and solutes in-between cells. Tight junctions consist of the transmembrane proteins occludin and claudin, and the cytoplasmic scaffolding proteins ZO-1, -2, and -3. This review discusses the binding interactions of the most studied proteins that occur within each of these two junctional complexes and possible modes of regulation of these interactions, and the different mechanisms that connect and regulate interactions with the actin cytoskeleton.     

Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions

Oleic acid (OA) affects assembly of gap junctions in neonatal cardiomyocytes. Adherens junction (AJ) regulates the stability of gap junction integrity; however, the effect of OA on AJ remains largely unexplored. The distribution of N-cadherin and catenins at cell–cell junction was decreased by OA. OA induced activation of protein kinase C(PKC)-α and -? and Src family kinase, and all three kinases were involved in the oleic acid-induced disassembly of the adherens junction, since it was blocked by pretreatment with Gö6976 (a PKCα inhibitor), ?V1–2 (a PKC? inhibitor), or PP2 (a Src family kinase inhibitor). Src family kinase appeared to be the downstream of PKC-α and -?, as blockade of either PKC-α or -? activity prevented the OA-induced activation of Src family kinase. Immunoprecipitation analyses showed that OA activated Fyn and Fer. OA promoted the association of p120 catenin/β-catenin with Fyn and Fer and caused increased tyrosine phosphorylation of p120 catenin and β-catenin, resulting in decreased binding of the former to N-cadherin and of the latter to α-catenin. Pretreatment with PP2 abrogated this OA-induced tyrosine phosphorylation of p120 catenin and β-catenin and restored the association of N-cadherin with p120 catenin and that of β-catenin with α-catenin. In conclusion, these results show that OA activates the PKC-Fyn signaling pathway, leading to the disassembly of the AJ. Therefore, inhibitors of PKC-α/-? and Src family kinase are potential candidates as cardioprotection agents against OA-induced heart injury during ischemia-reperfusion.     

Dynamic cross-talk between cells and the extracellular matrix in the testis

In the seminiferous tubule of the mammalian testis, one type A1 spermatogonium (diploid, 2n) divides and differentiates into 256 spermatozoa (haploid, n) during spermatogenesis. To complete spermatogenesis and produce approximately 150 x 10(6) spermatozoa each day in a healthy man, germ cells must migrate progressively across the seminiferous epithelium yet remain attach to the nourishing Sertoli cells. This active cell migration process involves precisely controlled restructuring events at the tight (TJ) and anchoring junctions at the cell-cell interface. While the hormonal events that regulate spermatogenesis by follicle-stimulating hormone and testosterone from the pituitary gland and Leydig cells, respectively, are known, less is known about the mechanism(s) that regulates junction restructuring during germ cell movement in the seminiferous epithelium. The relative position of tight (TJs) and anchoring junctions in the testis is of interest. Sertoli cell TJs that constitute the blood-testis barrier (BTB) are present side by side with anchoring junctions and are adjacent to the basement membrane. This intimate physical association with the TJs, the anchoring junctions and the basement membrane (a modified form of extracellular matrix, ECM) suggests a role for the ECM in the junction dynamics of the testis. Indeed, evidence is accumulating that ECM proteins are crucial to Sertoli cell TJ dynamics. In this review, we discuss the pivotal role of tumor necrosis factor alpha (TNFalpha) on BTB dynamics via its effects on the homeostasis of ECM proteins. In addition, discussion will also be focused on the novel findings regarding the role of non-basement-membrane-associated ECM proteins and components of focal adhesion (a cell-matrix anchoring junction type) in the regulation of junction dynamics in the testis.     

Mechanics of the Formation,Interaction, and Evolution of Membrane Tubular Structures

Membrane nanotubes, also known as membrane tethers, play important functional roles in many cellular processes, such as trafficking and signaling. Although considerable progresses have been made in understanding the physics regulating the mechanical behaviors of individual membrane nanotubes, relatively little is known about the formation of multiple membrane nanotubes due to the rapid occurring process involving strong cooperative effects and complex configurational transitions. By exerting a pair of external extraction upon two separate membrane regions, here, we combine molecular dynamics simulations and theoretical analysis to investigate how the membrane nanotube formation and pulling behaviors are regulated by the separation between the pulling forces and how the membrane protrusions interact with each other. As the force separation increases, different membrane configurations are observed, including an individual tubular protrusion, a relatively less deformed protrusion with two nanotubes on its top forming a V shape, a Y-shaped configuration through nanotube coalescence via a zipper-like mechanism, and two weakly interacting tubular protrusions. The energy profile as a function of the separation is determined. Moreover, the directional flow of lipid molecules accompanying the membrane shape transition is analyzed. Our results provide new, to our knowledge, insights at a molecular level into the interaction between membrane protrusions and help in understanding the formation and evolution of intra- and intercellular membrane tubular networks involved in numerous cell activities.     

Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission

Transmission of HIV-1 via intercellular connections has been estimated as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.     

Role of β-catenin in post-meiotic male germ cell differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.     

Cutting edge: Membrane nanotubes connect immune cells

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.     

Vinculin and α-catenin: shared and unique functions in adherens junctions

Vinculin and α-catenin are two functionally related proteins of adherens junctions, structures in which cells make contacts to neighboring cells or to the extracellular matrix. At these sites, the actin cytoskeleton of animal cells is anchored to the plasma membrane. Junction assembly and disassembly are coordinated in processes as different as mitosis, cell movement and tissue formation. Since adherens junctions are assembled from a large number of proteins, these molecules have to be coordinately activated and spatially regulated. Vinculin and α-catenin have been characterized as tumor suppressors, suggesting that they have a regulatory function in addition to their structural role. Several possible modes of vinculin and α-catenin regulation are discussed here, as the published data favor the concept that no single model fully explains the complexity of adherens junctions. Most probably, cells select from a variety of possibilities to solve the problem of making specific contacts. BioEssays 20 :733–740, 1998.© 1998 John Wiley and Sons, Inc.     

Planar cell polarity protein localization in the secretory ameloblasts of rat incisors

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.