Cooperation of Nectin-1 and Nectin-3 Is Required for Maintenance of Epidermal Stratification and Proper Hair Shaft Formation in the Mouse

constitute a family of four cell adhesion molecules which are localized on cell membrane. Mutations in NECTIN-1 gene


Introduction
Nectins are immunoglobulin-(Ig-) like, calcium-independent cell adhesion molecules involved in various cellular and physiological processes including proliferation, migration, polarization, and adhesion [1]. The nectins are encoded by PVRL genes and the family is composed of four members, nectin-1 to nectin-4. Each nectin has an N-terminal extracellular domain with three Ig loops, a transmembrane domain, and a C-terminal intracellular domain. Nectins interact with nectins or other cell surface molecules on adjacent cells through the Ig loops. The nectin-nectin interactions are either homophilic or heterophilic, that is, interactions between the same or a different nectin, respectively [2]. The nectin interaction elicits intracellular signalling via the binding of the C-terminal domain to afadin [3]. Of the cellular functions regulated by nectins, cell adhesion has been extensively studied.
Nectin signalling recruits cadherins to the junctional site forming an adherens junction, and this is followed by the recruitment of claudins forming a tight junction [4][5][6]. Recent studies in mouse show that nectin interactions are involved in formation and/or maintenance of desmosomes as well [7,8]. The identification of mutations in NECTIN-1 (PVRL1) gene as the cause of severe human ectodermal dysplasia (ED) syndrome Cleft Lip/Palate-Ectodermal Dysplasia Syndrome 1 (CLPED1, MIM 225060) indicates an important role for nectin in the development of mammalian skin and its appendages [9,10]. In addition, during the course of our study mutations in NECTIN-4 were reported to cause ED [11]. EDs are congenital disorders characterized by abnormalities in two or more ectodermal organs such as teeth, hair, epidermis, 2 Developmental Biology Journal and several exocrine glands. The ectodermal organ defects in CLPED1 patients include kinky and sparse hair and thickening of palm skin [12][13][14]. The mutations identified in CLPED1 lead to truncated protein that lacks the transmembrane domain and C-terminus [9,15]. As the interaction of the C-terminus with afadin is required for signalling, the mutations are thought to cause the loss of nectin signalling and therefore, the defects of CLPED1 patients are thought to be caused by lack of nectin-1 signalling.
Mammalian skin is composed of epidermis, dermis, and hypodermis and its appendages include pelage hair. The epidermis forms the primary barrier against the external environment and prevents water loss, and pelage hair supports this function [16]. The epidermis comprises four distinct cell layers: the basal, spinous, granular, and cornified layers [17]. The basal cells are attached to the basement membrane and proliferate to supply cells for the turnover of epidermis. During epidermal stratification epithelial cells differentiate progressively and express specific proteins for each cell layer, eventually forming the denucleated and keratinized cornified envelope [16]. Pelage hair is formed by hair follicles composed of a dermal papilla which is surrounded by epithelial tissue including matrix and the inner and outer root sheath (IRS and ORS, resp.) [18]. The matrix cells proliferate actively and give rise to IRS cells and hair shaft. IRS is composed of several layers and supports the hair shaft towards the surface of epidermis. Both in the epidermis and hair follicle, epithelial cells are connected via cell-cell junctions including adherens and tight junctions and desmosomes [19][20][21]. Previous studies demonstrated that mutant mice exhibiting modulation of cell adhesion and loss of junctional proteins show abnormal differentiation and structure of both epidermis and hair follicles [22]. These reports indicate the importance of cell-cell junctions for skin development and homeostasis.
During mouse development, nectins are expressed in ectodermal tissues including epidermis and hair follicle [8,23,24]. Nectin-1, nectin-2, and nectin-3 have been knocked out in mice, but none of the single mutant mouse lines exhibits severe abnormalities in ectodermal organs. Neither epidermis nor hair abnormalities have been reported in nectin-2 null mutants [25,26]. Nectin-3 null mutants exhibit mild defects in dental enamel, eye, and neurogenesis [8,[27][28][29]. Intriguingly, although CLPED1 patients exhibiting mutations in NECTIN-1 gene manifest severe aberrations in hair and tooth development, nectin-1 deficient mice have only mild defects in skin including fragile epidermis with reduced expression of the granular layer marker loricrin, and minor defects in enamel [7,8,30]. In addition, nectin-1 null mutants show mild defects in the eye and neurogenesis [27,29]. Hence, nectin-1 null mutant mice do not recapitulate the phenotypes of human CLPED1 and they cannot be used as an appropriate animal model to examine the pathogenesis of CLPED1 or the role of nectins in the morphogenesis of ectodermal organs. As previous studies have reported that overlapping expression of nectins in skin and nectins may act via heterophilic nectin-nectin interactions, we hypothesized that the function of nectin-1 may be compensated by other nectins in mice [8,24]. As the interaction between nectin-1 and nectin-3 is the strongest among nectin interactions [2] and nectin-1 and nectin-3 mutant mice have overlapping abnormalities, nectin-3 appeared as a good candidate for the compensating nectin.
We generated compound mutant mice of nectin-1 and nectin-3 [8] and observed more severe skin defects than in single null mutants. The epidermis of compound mutant mice exhibited a cornification defect and abnormalities in the basal epithelial layer, and the structure of the pelage hair was aberrant. The expressions and localization of cell adhesion molecules associated with adherens and tight junctions and desmosomes were severely impaired in the compound mutant epidermis and hair follicles. These abnormalities were not observed during embryonic development. Our results indicated that cooperation of nectin-1 and nectin-3 is important for postnatal development and homeostasis of mouse epidermis and pelage hair.

Animals.
Wild-type NMRI mice were used for gene expression analysis. The nectin-1 and nectin-3 single mutants were in the C57BL/6 background and double heterozygotes were generated by crossing them [29]. Compound mutants were generated by crossing the male and female double heterozygotes as previously described [8]. The appearance of a vaginal plug was taken as embryonic day 0.

Preparation of Tissues and Histology.
Skin from mouse pups and embryos was dissected, fixed in 4% paraformaldehyde, and processed for paraffin sections. The sections were cut at 5 m and used for haematoxylin and eosin (HE) staining, in situ hybridization, and immunohistochemistry.

Expression Patterns of Nectin-1 and Nectin-3 in Epidermis and Hair
Follicle. We first investigated the expression of nectins in the embryonic and postnatal skin. Since the expression of nectin-3 could not be detected by immunohistochemistry, we used radioactive in situ hybridization (ISH) in this study. Previous studies have reported that nectin-1 is expressed in the suprabasal layer of epidermis and in all cells of the whisker follicle except for outer root sheath (ORS) [23,24,30], while no obvious expression of nectin-3 was observed in these tissues [24]. Consistent with these previous studies we observed nectin-1 expression in the spinous and granular layers of epidermis and hair follicle at embryonic day 17 (E17), while no specific signal for nectin-3 was detected (see Supplementary Figure S1

The Macroscopic Phenotype of Nectin-1 and Nectin-3 Compound Mutant Mice.
Nectin-1 and nectin-3 compound mutants were generated by crossing double heterozygote mice as previously described [8]. Until postnatal day 5, no obvious difference was detected in the overall phenotype of the pups. Thereafter, the growth of compound mutants was retarded and sparse hair was observed in all compound mutants (nectin-1−/−; nectin-3+/− ( = 5), nectin-1+/−; nectin-3−/− ( = 4), and nectin-1−/−; nectin-3−/− ( = 1), all these genotypes were called "compound mutant"), but not in wild types, single, or double heterozygote mice (all these genotypes were used as controls). Due to poor health, the compound mutant mice had to be sacrificed at P10. There was significant phenotypic variation among the compound mutant mice with regard to appearance of the hair, but interestingly, the severity of the skin phenotype did not correlate with specific compound genotypes. Only one double KO (nectin-1−/−; nectin-3−/−) mouse was available for analysis at P10 and it exhibited a similar, rather mild skin abnormality in histological and immunohistochemical analysis as compared with some of the nectin-1−/−; nectin-3+/− mice (see supplementary Figure S3). The phenotypes of nectin-1+/−; nectin-3−/− mice were comparable to those of nectin-1−/−; nectin-3+/− (see Figure 3). We mainly describe the phenotype of nectin-1−/−; nectin-3+/− mice in this report. At P10, the compound mutants had variable deviations in body size (Figures 1(g)-1(i)) and hair thickness. The long guard hairs visible in macroscopical examination of controls (arrowhead in Figure 1(g )) were not observed in the mutants. However, they were actually present as shown by microscopy (see below). Obvious hair abnormalities were observed also in ventral skin in several compound mutant mice (Supplementary Figure S2).

The Compound Mutant Epidermis Exhibits Stratification Defects, Abnormal Basal Cell Layer, and Junctional Defects.
During the dissection of the skin and its processing for histology, their skin sections detached readily from the glass slides during sectioning and staining procedures. In histological analysis, the compound mutant skin detached between dermis and subcutis (Figures 2(a), 2(b), 2(f), and 2(g)).
Higher magnifications revealed morphological abnormality of basal cell nuclei and epidermal layers in the compound mutant epidermis (Figures 2(b), 2(c), 2(g), and 2(h)). Hair follicles of the compound mutants were hypoplastic and exhibited abnormal structure compared with that of the controls (Figures 2(d), 2(e), 2(i), and 2(j)). Notably, the nuclei of the compound mutant hair follicles could be observed only after prolonged haematoxylin staining or nuclear staining using DAPI (inset of Figure 2(j) and see Figure 5(l )).
Next we addressed the differentiation of the compound mutant epidermis using immunohistochemical analysis of markers for each epidermal cell layer. The following antibodies against proteins specific for basal, spinous, granular, and cornified layers were used for immunostaining: keratin 14 (K14), K10, loricrin, and filaggrin, respectively [17]. Loricrin expression was previously shown to be reduced in nectin-1 single mutants, while expressions of the markers for other cell layers were indistinguishable from controls [30]. In the compound mutants, the expressions of K14, loricrin, and filaggrin as well as basement membrane protein laminin were severely affected, while the expression of spinous layer marker K10 was observed in upper layers (Figures 3(a)-3(i), and 3(l)). In the basal layer, the expressions of cell proliferation marker Ki67 and basal layer marker p63 were almost lost in some compound mutants (Figures 3(j), 3(k), 3(m), and 3(n)), while in some mutants residual expression was apparent (Figures 3(o) and 3(p)). K14 is a known target of p63 [32] and in line with this, the triple immunostaining indicated that their levels of expression correlated in individual cells (Figures 3(o) and 3(p), Supplementary Figure S3). In conclusion, our observations indicated that the stratification of the epidermis and the proliferation and morphology of cells in the basal layer were affected by reduced nectin expression.
Previous studies have reported that nectin-nectin interactions recruit adherens and tight junctions [2] and desmosomes [7] and that disturbed cell adhesion results in abnormal differentiation of the epidermis [33][34][35]. We therefore investigated the expression of different junctional proteins to clarify the pathogenesis of the observed defects in epidermis. Adherens junctions and desmosomes form throughout epidermis, while tight junctions mainly form between granular layer cells [36,37]. We used antibodies against E-cadherin and ZO-1 to detect adherens and tight junctions, respectively. Desmosomes were detected by an antibody recognising both desmoglein 1 and 2 (desmoglein 1/2). As desmoglein 1 is present in the suprabasal layers and desmoglein 2 in the basal layer [38] the antibody detects desmosomes throughout epidermis. The epidermis of the compound mutants exhibited impaired expression and localization of these marker proteins of the adherens and tight junctions and desmosomes (Figures 4(a)-4(f)). It is likely that the observed defects in the epidermal structure and cell adhesion contributed to the weakness of the skin and the poor general condition of the mice.

The Hair Follicles and Hair Shafts of the Compound
Mutants Exhibit Disturbed Structure and Impaired Cell Adhesion. The pelage hair of the compound mutants exhibited * * * * * * *

Nectin-3
Sense control Nectin-1  severe abnormalities. We first analysed the structure and types of hair plucked from mice of controls and all compound mutant genotypes at P10. Analysis of the plucked hair under stereomicroscope indicated that comparable hair types to the control (guard, awl, and zigzag) were present in all compound mutants (data not shown). This indicates that the long guard hair had developed in the mutants although they were not observed in the macroscopic examination of the mice (Figures 1(h ) and 1(i )). Higher magnification revealed, however, that the air cells in the guard hair shafts of the compound mutants exhibited disorganized patterns (Figures 5(a) and 5(d)). A similar air cell abnormality was also observed in the other hair types (data not shown). The expression pattern of K17 in hair shafts [39] and nuclear staining using DAPI also showed abnormal localization of the hair matrix protein and arrangement of nuclei, respectively (Figures 5(b) and 5(e)). Abnormal distribution of K17 immunoreactivity and lack of laminin expression in ORS [40,41] also revealed an abnormal structure of ORS ( Figures 5(b), 5(c), 5(e), and 5(f)). We next localized cell adhesion molecules to investigate the formation of cellular junctions in the abnormal hair shafts and hair follicles of the compound mutants. Previous studies show that E-cadherin localizes to the adherens junctions between IRS and ORS [42] and that the desmosomal and tight junction proteins desmoglein 1/2 and ZO-1 are present in IRS, ORS, and matrix in hair follicles [37]. Although we observed the expression of the markers of all three types of junctions in the hair follicles of compound mutants, the localizations of these junctional proteins were diffuse, and no obvious membrane localization was detected (Figures 5(g)-5(l)). We did not observe severe loss of Ki67 and p63 expression in the compound mutant hair follicles (Supplementary Figure  S4). This suggested that the hair follicle and hair shaft abnormalities were caused by the defects in cell adhesion due to the deficiency of nectin-1 and nectin-3.

No Significant Skin Abnormalities Were Observed in the
Embryos of Compound Mutants. The embryonic epidermal and hair follicle phenotypes of the compound mutants were investigated at E14 and E17. At E14, the external appearance of the skin of the mutant embryos was indistinguishable from the controls (Supplementary Figure S5) and histological examination of the epidermis and hair follicles did not reveal abnormalities either in cell and tissue morphology or in protein localization (Supplementary Figure S5). Notably, the placodes of the first wave of hair follicles giving rise to guard hair were apparent in the E14 compound mutants with similar patterns to those in the control embryos. The localization of E-cadherin in E14 hair placodes and whisker follicles was normal in the compound mutants (Supplementary Figure  S5.). At E17, the mutant embryos appeared to be similar to the controls when observed under stereomicroscope (Supplementary Figure S6). In histological sections, morphology of E17 compound mutant epidermis was similar to controls, and the follicles of guard hair as well as awl hair were present both in the compound mutants and controls with indistinguishable histological features (Figures 6(a) and 6(f)). Immunohistochemical analysis revealed that the basal and cornified cell layer markers were slightly reduced in intensity in the compound mutant epidermis (Figures 6(b), 6(c), 6(g), and 6(h)). However, no obvious abnormalities were observed either in the expression or in the localization of adherens junction and desmosomal proteins in the compound mutant epidermis and hair follicle in the embryos (Figures 6(d),   6(h), 6(i), and 6(l)). The expression of Ki67 and p63 in the basal layer cells of the E17 compound mutant epidermis was also comparable to the control (Figures 6(j), 6(k), 6(m), and 6(n)). These results suggested that the epidermal and hair phenotypes of the compound mutants become apparent mainly postnatally.

Nectin-1 and Nectin-3 Are Required for Cell Adhesion and Differentiation in the Epidermis and Hair
Follicle. The compound mutant epidermis was fragile, and the severely reduced or absent expression of filaggrin and loricrin indicated defects in the granular and cornified cell layers.
The adherens and tight junctions as well as desmosomes were all severely impaired as shown by the dispersed or completely absent expression of their specific markers and abnormal ultrastructure. Mice lacking the tight junction protein claudin-1 exhibit barrier defect in skin and die because of excess water loss [43], while mutant mice that lack components of other junctions in epidermis survive longer [33,35,44,45]. Therefore, it is possible that defective tight junctions in the nectin compound mutants are the primary cause of the compromised fitness and fragility of their skin. Since nectin interaction recruits tight junctions through adherens junction formation [1], the cornification defects in the nectin mutants may be secondary to the lack of adherens junctions, and in line with this, a mouse strain lacking E-cadherin in skin also lacks tight junctions [34].
Interestingly, similar to the nectin mutants reported here, the mice lacking E-cadherin in epidermis exhibit reduction of loricrin and filaggrin but retain the expression of K10 in the suprabasal cells [33,35]. We observed abnormal cell shapes and intracellular structures in light and electron microscopy of epidermal cells in the nectin compound mutants. Similar observations were reported in mice lacking intermediate filament proteins K14 and K17 [46,47] and it is known that a desmosome is connected to intermediate filaments through desmoplakin [38]. We observed diffuse localization of desmoglein 1/2 throughout the epidermis and abnormal desmosomes between cells of the basal and suprabasal layers in the compound mutants. We hypothesized that nectin deficiency in the suprabasal cells caused the defects in desmosomes, which was followed by abnormal organization of intermediate filaments resulting in the abnormal ultrastructure of epidermal cells.
Interestingly, the compound mutant epidermis exhibited severe defects also in the basal layer although expression of neither nectin-1 nor nectin-3 was detected in these cells. The shapes of basal cell nuclei were abnormal and the expression of laminin, K14, and p63, all required for the maintenance and proliferation of basal layer cells [47][48][49], was reduced. In addition, expression of E-cadherin and desmoglein 1/2 was impaired. As desmosomes affect cell shape and elicit intracellular signalling involved in cellular viability [50,51], it is conceivable that the defects in the basal cells were secondary to the nectin deficiency in the spinous layer and may have resulted from compromised function of desmosomes.  The immunohistochemical observation of hair follicles indicated similar defects in cellular junctions and intracellular structures as in epidermis, and the aberrant hematoxylin and eosin (HE) staining of hair matrix cells conceivably resulted from these structural defects. The hair follicles of the compound mutants were hypoplastic and the hair shafts were structurally abnormal as indicated by disorganized air cells and disturbed expression of the hair shaft protein K17. This may have contributed to reduced stiffness of the hair and to our failure to observe long guard hair in macroscopical examination of mutant fur. Since the deficiency of adherens junctions and desmosomes results in abnormal structure of the hair follicle and hair shaft [33,35,44], it is likely that the similar phenotypes seen in the nectin mutants were caused by impaired cell adhesion.

Heterophilic Interaction between Nectin-1 and Nectin-3 Is Required for Recruitment of Cell-Cell Junctions in Postnatal
Epidermis and Hair Follicles. The phenotypes of the compound mutants were significantly more severe than those of nectin-1 or nectin-3 single mutants, indicating cooperative roles of these two nectins. The defects in epidermis and hair exhibited marked variation between the mice of the same genotype, and interestingly, we did not detect obvious differences between nectin-1−/−; nectin-3+/− and nectin-1+/−; nectin-3−/− genotypes. The interaction between nectin-1 and nectin-3 is the strongest among homophilic and heterophilic nectin interactions, but nectin-1 and nectin-3 also interact with nectin-4 and nectin-2 which are expressed in postnatal and embryonic skin [1]. Taken together, our results indicated that, in single nectin-1 and nectin-3 null mutant mice, homophilic interaction of the remaining nectin (i.e., nectin-3 in nectin-1−/− and vice versa) partially compensated for the function of the lacking nectin. Additional loss of one copy of the remaining nectin reduced the compensatory interaction and was sufficient to cause the severe epidermal and hair abnormalities. We therefore suggested that the intensity of nectin signalling, reflecting the amount and strength of nectin interactions, was critical for recruitment of cell-cell junctions in epidermis and hair follicle. Of specific interest was the localization of nectin-1 and nectin-3 in adjacent cell layers and the IRS and ORS in the hair follicle. This presents an example of heterophilic nectin interactions between adjacent cell layers required for proper development in various organs [8,29,52,53]. The compound mutant epidermis exhibited more severe abnormalities than in hair follicle. The compound mutant epidermis exhibited severely reduced expression of Ki67 and p63, while only slight reductions of p63 and Ki67 expression were observed in the compound mutant hair follicle. This might be explained by the different expression pattern of nectin-1 and -3 in epidermis and hair follicle. In the epidermis, both nectin-1 and nectin-3 were expressed in suprabasal layer. In hair follicle, nectin-1 was expressed in IRS and nectin-3 was expressed in ORS. Thus, the loss of nectin-1 and -3 was thought to cause more severe loss of signal from suprabasal layer to basal layer than that from ORS to IRS. This might lead to the more severe abnormalities in epidermis than that in hair follicle.
Although the overall health of the compound mutants was compromised at P10 and they had severe skin defects, the pups appeared macroscopically normal at P5 and no significant abnormalities were observed in the epidermis and hair follicles during embryogenesis. This was consistent with the results that nectin-1 mutants have no apparent embryonic phenotype and that nectin-3 appeared not to be expressed in the embryonic skin. However, as nectin-2 and nectin-4 are expressed in the embryonic skin [24], it is likely that they further compensate for nectin function during embryonic development.
In conclusion, our results suggest that heterophilic interaction between nectin-1 and nectin-3 is required for postnatal epidermal development and proper pelage hair formation in mice. In particular, nectin deficiency affected adherens, desmosomal, and tight junctions and reduced indirectly basal cell proliferation, thereby leading to severe skin phenotype. These findings increase the understanding of the role of nectins in epidermis and hair development.