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Nectins Establish a Checkerboard-Like Cellular Pattern in the Auditory Epithelium

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Science  26 Aug 2011:
Vol. 333, Issue 6046, pp. 1144-1147
DOI: 10.1126/science.1208467

Abstract

In the auditory epithelium of the cochlea, the sensory hair cells and supporting cells are arranged in a checkerboard-like fashion, but the mechanism underlying this cellular patterning is unclear. We found that mouse hair cells and supporting cells express the immunoglobulin-like adhesion molecules nectin-1 and -3, respectively, and that their interaction mediates the heterotypic adhesion between these two cell types. Genetic removal of nectin-1 or -3 disrupted the checkerboard-like pattern, inducing aberrant attachment between hair cells. When cells expressing either nectin-1 or -3 were cocultured, they arranged themselves into a mosaic pattern. Thus, nectin-1 and -3 promote the formation of the checkerboard-like pattern of the auditory epithelia.

Animal tissues comprise multiple cell types, which are arranged in complex, elaborate patterns. One such pattern is the checkerboard-like cell arrangement, which is observed in certain tissues such as the oviduct and the auditory epithelia (1, 2). The organ of Corti in the inner ear consists of mechanosensory hair cells (subgrouped into inner and outer hair cells), which are interdigitated with various types of supporting cells (2); these cells are arranged in a checkerboard-like pattern (fig. S1). A number of genes or molecules, including those known to regulate planar cell polarity, have been implicated in the generation of the highly ordered structures in the cochlea (37). A mathematical model predicted that the checkerboard-like pattern could be generated by a mixture of two cell types, when their heterotypic cell-cell adhesions dominated over their homotypic ones (8). However, how the checkerboard-like pattern of cells is established is unclear.

Epithelial cell-cell adhesions are mediated via the adherens junctions (AJs), where two classes of molecules, cadherins and nectins, cooperate. The major role of cadherins is to connect cells expressing the same cadherins through their homophilic interactions (9, 10). In contrast, nectins, a family of immunoglobulin-like molecules, comprising four members (nectin-1, -2, -3, and -4), promote both homophilic and heterophilic interactions between the members (1113). Their heterophilic interactions are stronger than their homophilic interactions in the following order: nectin 1-3 > nectin 2-3 > nectin 1-1, 2-2, and 3-3, which might contribute to the mosaic patterning of two cell populations (1116). We investigated the potential role of nectins in the formation of the checkerboard-like pattern of the auditory epithelial cells in the mouse.

We first examined the localization of nectin-1, -2, and -3 in the auditory epithelia at different stages of development. At embryonic day 14 (E14), the three nectins were already localized at the junctions between the auditory epithelia, although the checkerboard-like cellular pattern was not clearly visible; this pattern became detectable at E16 and even clearer at postnatal day 1 (P1). In the middle turn of the cochlea at P1, nectin-1 was localized only at the heterotypic boundaries between hair (myosin VIIa–positive) and supporting (myosin VIIa–negative) cells (Fig. 1, arrows), not at the junctions between supporting cells. The distribution of nectin-3 was similar to that of nectin-1; that is, nectin-3 was localized along the heterotypic junctions between hair cells and supporting cells (Fig. 1, arrows). However, unlike nectin-1, nectin-3 was also weakly detected at the junctions between supporting cells (Fig. 1, arrowheads). On the other hand, nectin-2 was uniformly distributed at the boundaries between hair cells and supporting cells, as well as between supporting cells (Fig. 1). The localization patterns of nectins in the auditory epithelia at E16 were essentially identical to those at P1 (fig. S2). All of these nectin molecules were located on the apical side of the lateral membranes of hair cells and supporting cells, where the AJs are detected (2, 17) (fig. S3). To identify the cells expressing nectin-1 and -3, we performed in situ hybridization for each at P1. Nectin-1 mRNA was expressed in hair cells, whereas nectin-3 mRNA was detected in supporting cells (fig. S4). Thus, nectin-1 expressed in hair cells and nectin-3 expressed in supporting cells are concentrated at their heterotypic boundaries (fig. S1E).

Fig. 1

Localization of nectins in the auditory epithelia of wild-type mice at P1. Nectins and myosin VIIa (MyoVIIa) were doubly stained at apical surfaces of the auditory epithelia in the middle turn of cochleae. Hair cells were identified on the basis of MyoVIIa expression. OHCs, outer hair cells; IHCs, inner hair cells. Arrows point to examples of nectin-1 or -3 signals concentrated at the borders between hair cells and supporting cells. Arrowheads indicate examples of nectin-3 signals concentrated at junctions between supporting cells.

We next analyzed the cochleas of mice in which the genes encoding these nectins were knocked out (KO) (Fig. 2). The numbers of differentiated hair cells and supporting cells were not altered in these mutant mice (Fig. 2F). However, the checkerboard-like pattern of these cells was dramatically affected by the deficiency of nectin-3. In nectin-3 KO mice, two or three hair cells aberrantly attached to each other, as revealed by staining for F-actin and the tight-junction marker ZO-1 (Fig. 2D, arrows). The signals for ZO-1 and F-actin appeared as a single line at the borders between neighboring hair cells, indicating that these cells came into direct contact with each other. Most (62.4%, n = 15) outer hair cells aggregated in this manner, disrupting the checkerboard-like pattern. Inner hair cells were also aberrantly attached to one another, although less frequently than outer hair cells. In addition, these inner hair cells lost their contacts with inner pillar cells, resulting in the elongation of the contact sites between inner phalangeal cells (depicted in gray in fig. S1D) (Fig. 2D, arrowheads). In nectin-1 KO mice, similar phenotypes were observed, but the effects of nectin-1 KO were much milder than those of nectin-3 KO in mice (Fig. 2, B and E). On the other hand, nectin-2 KO mice did not show any detectable phenotypes (Fig. 2C).

Fig. 2

Cellular patterning in the auditory epithelia of wild-type (WT) and nectin KO mice at P1. (A to D) F-actin and ZO-1 were doubly stained in the middle turn of the cochlea. Arrows point to examples of contact sites aberrantly formed between hair cells. Arrowheads indicate abnormally formed junctions between inner phalangeal cells. (E and F) Statistical analysis of nectin KO phenotypes. (E) Number of hair cells showing aberrant attachment to other cells in each photographic field, the size of which is equivalent to those in (A) to (D). Left, attached OHCs. Middle, attached IHCs. Right, IHCs detached from inner pillar cells (PC). (F) Numbers of supporting cells (SC), OHCs, IHCs, and total hair cells (HC) per photographic field. Results shown are the means ± SEM. *P < 0.01; **P < 0.001. Data were collected from three individuals and were quantified using five fields in each individual.

To understand how the above phenotypes were generated, we examined the distribution patterns of the nectins remaining in these KO mice (Fig. 3). In nectin-1 KO mice, the uniform distribution of nectin-2 was not altered (Fig. 3B), whereas nectin-3 became localized more intensely at the junctions between supporting cells (Fig. 3B, arrowheads). In nectin-2 KO mice, the localization patterns of nectin-1 and -3 remained unchanged (Fig. 3C). In nectin-3 KO mice, nectin-1 was lost from the heterotypic junctions between hair cells and supporting cells; instead, it became concentrated at the boundaries between aberrantly contacting hair cells (Fig. 3D, arrows, and fig. S5), suggesting that the nectin-1 in these mice was undergoing only homophilic interactions at the interfaces between hair cells. Thus, the loss of nectin-1 and -3 caused the redistribution of their heterophilic partners to ectopic sites.

Fig. 3

(A to D) Distribution of nectins in the middle turn of cochleae of wild-type and nectin KO mice at P1. Arrowheads point to examples of up-regulated nectin-3 signals at the boundaries between supporting cells. Arrows point to nectin-1 signals abnormally concentrated at the boundaries between hair cells.

To verify the roles of nectin-1 and -3 in cellular patterning, we reexamined the behavior of cells expressing these nectins in cultures. We prepared HEK293 cells stably expressing nectin-1 or -3, into which the fluorescent proteins enhanced green fluorescent protein (EGFP) or mCherry were introduced to distinguish between the two cell lines. These cells were sparsely cultured to allow the formation of independent colonies. When their colony edges came into contact with one another, their boundaries were examined. Cells expressing identical nectin types did not intermingle at the border, whereas those expressing nectin-1 and -3 mutually invaded the counter colony, resulting in the formation of a mosaic pattern (Fig. 4, A to E, and fig. S6). We also performed time-lapse video microscopy using a coculture of MDCK cells expressing nectin-1 or -3 (N1- and N3-MDCK cells). In the supporting movie (movie S1 and Fig. 4E), one N1-MDCK cell (arrowhead) initially adhered to one of a pair of N3-MDCK cells (asterisks); subsequently, the former cell invaded the space between the two N3-MDCK cells. As a result, N1- and N3-MDCK cells were rearranged into a mosaic pattern. Similar behavior of cells was repeatedly observed in multiple experiments.

Fig. 4

Mosaic patterning of cells formed by heterophilic interaction of nectin-1 and -3. (A to D) Boundaries between two colonies of HEK293 cells, which were doubly transfected with a nectin and EGFP (green) or mCherry (red) in the combinations indicated. (A) Control cells expressing EGFP and mCherry; (B) cells expressing nectin-1/EGFP and nectin-1/mCherry; (C) cells expressing nectin-3/EGFP and nectin-3/mCherry; (D) cells expressing nectin-1/EGFP and nectin-3/mCherry. (E) Time-lapse microscopy of a coculture of MDCK cells expressing nectin-1/EGFP and those expressing nectin-3/mCherry. Arrowhead, cell expressing nectin-1; asterisks, pair of cells expressing nectin-3. Time (t) is in minutes. (F) Hypothetical profiles of nectin-nectin interactions at various cell-cell boundaries. Bidirectional arrows indicate heterophilic or homophilic interactions between nectins. The thickness of the arrows represents the relative strength of nectin-nectin interactions. The broken arrow indicates less frequent cases. When heterophilic interaction between nectin-1 and -3 occur, the majority of these molecules are recruited to heterophilic binding sites; therefore, their potential homophilic interactions are not depicted in the wild-type cartoon. This may also be the case for interactions between nectin-2 and -3, but this has not been tested.

Thus, we propose that the heterophilic interactions between nectin-1 and -3 are critical for establishing the checkerboard-like pattern of hair cells and supporting cells. The molecular interaction between nectin-1 and -3 is the strongest of all possible combinations of the three nectins, which is likely to be responsible for the checkerboard-like assembly of these cells (Fig. 4F), as predicted by the mathematical model (8). The loss of nectin-3 removed such biased cell-cell adhesion, leading to cell rearrangement, including attachments between hair cells (Fig. 2D), as explained by the differential adhesiveness hypothesis (18). Nectin-1 KO mice displayed milder phenotypes. In these mice, the relatively strong interaction between nectin-3 and -2 probably retained the adhesion between hair cells and supporting cells; on the other hand, the adhesion between supporting cells should have been enhanced as a result of the redistribution of nectin-3 to these sites. These combinatory situations probably suppressed adhesion between hair cells (Fig. 4F). In nectin-2 KO mice, the heterophilic interactions between nectin-1 and -3 persisted; this explains the absence of a phenotype in these mice. In the absence of nectins, the cell junctions were not disrupted. This is most likely due to the coexpression of classic cadherins in the auditory epithelia. Hair cells and supporting cells are thought to be segregated through the process of lateral inhibition mediated by Notch-Delta signaling (4, 19), and such processes themselves might contribute to the spatial separation of these cells (2022). However, genetic inactivation of Notch signaling does not impair the checkerboard-like pattern, although it does result in an increase in the number of hair cells (4). This suggests that lateral inhibition is insufficient to create the checkerboard-like cellular pattern, stressing the importance of nectins in this patterning process. It is of note that heterophilic interactions between Hibris and Roughest, other members of the immunoglobulin superfamily, also contribute to the cell arrangement in the Drosophila eye (23, 24), suggesting that similar mechanisms are conserved for cellular patterning across species.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1208467/DC1

Materials and Methods

Figs. S1 to S6

References (2527)

Movie S1

References and Notes

  1. Acknowledgments: This work was supported by the Global Centers of Excellence Program “Global Center for Education and Research in Integrative Membrane Biology” and the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology in Japan; by Grants-in-Aid from the Japan Society for the Promotion of Science; and by the Core Research for Evolutional Science and Technology from the Japanese Science and Technology Agency.
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