Multi-ciliated cells line the surface of the oviduct, the trachea, and the ventricle of the brain (Figure 1A,B show oviduct multi-ciliated cells). At the apical surface of multi-ciliated cells, tens to hundreds of motile cilia are formed. The directions of cilia movements are unidirectionally aligned within each cell (rotational polarity), and they are consistent with the orientation of the tissue axis (tissue-level polarity). In addition, cilia exhibit a metachronal wave, a wave-like propagation of cilia movements in the plane of the tissue, which is produced by a temporal coordination of cilia movements between neighboring cilia (Figure 1C–D’) ^{[1][2][3][4][5]}[1,2,3,4,5]. The coordination of cilia movements is essential for the functions of organs. In the mammalian oviduct, cilia pick up ovulated oocytes at the ovary end of the oviduct and carry them to the uterus ^[6][7][6,7]; in the trachea, cilia transport mucus and eliminate debris and pathogens ^[8][9][8,9]; and in the ventricle, cilia generate a flow of cerebrospinal fluid that is required for homeostasis of the organ [10].

Motile cilia of multi-ciliated cells are microtubule-based cell protrusions which show a biphasic movement comprised of a fast effective stroke, and a slow backward motion, the recovery stroke (Figure 1D). Inside the majority of motile cilia, 9 + 2 arrays of microtubules run along the longitudinal axis of the cilium, and a central pair of microtubules lie perpendicular to the beating orientation of the cilium (Figure 1E,F) [11]. The basal body lies at the base of cilium, and the basal foot and the rootlet are associated with the basal body (Figure 1E). A single basal foot protrudes from the lateral side of each basal body, and its direction is consistent with that of the effective stroke ^[12][13][12,13]. A rootlet is located at the proximal end of each basal body and extends to the center of the cell ^{[14][15][16][17]}[14,15,16,17].

2. Roles of Core PCP Proteins in Coordinating Cilia Orientation

As exemplified by the unidirectionally beating cilia of multi-ciliated cells, various epithelial cells polarize not only along the apical-basal axis (inside–outside axis) of epithelial tissues, but also on the plane of epithelial tissues. The latter cell polarity, which is perpendicular to the apical–basal axis, is referred to as planar cell polarity (PCP) ^{[18][19][20][21][22][23][24][25][26][27][28]}[18,19,20,21,22,23,24,25,26,27,28]. Pioneering research using the wings of Drosophila melanogaster identified a group of proteins, core PCP proteins, that orchestrate the establishment of PCP (Figure 2). Core PCP proteins are an evolutionally conserved group of proteins comprised of transmembrane proteins, Flamingo/Starry night (Fmi/Stan), Van Gogh (Vang) and Frizzled (Fz), as well as cytoplasmic proteins Prickle (Pk), Dishevelled (Dvl) and Diego (Dgo) ^{[21][26][29][30]}[21,26,29,30]. Each Drosophila wing epithelial cell forms an actin-rich cell protrusion, a wing hair, at the apical cortex and each wing hair points to the distal end of the wing, which is a hallmark of PCP in the wing [21]. Just before the onset of wing hair formation, cell boundary localization of core PCP proteins is strongly biased along the tissue axis (Figure 2A). Fz- and Vang-containing complexes (referred to as the Fz- and Vang-complex in Drosophila, respectively, or the FZD- and VANGL-complex in vertebrates, as used hereafter) localize at the distal and proximal side of the cell, respectively (Figure 2A,A’) ^{[28][31][32][33][34][35]}[28,31,32,33,34,35]. When each member of core PCP proteins is lacking, the orientations of wing hairs are not coordinated along the tissue axis [21]. In addition to the Drosophila wing, the asymmetric distribution of core PCP proteins was observed in various organs and animals, including multi-ciliated cells of the mouse oviduct, trachea, and ventricle (Figure 2B) ^{[20][36][37][38]}[20,36,37,38]. The establishment of the polarized distribution of core PCP proteins precedes the formation of multi-cilia in the developing oviduct ^[20][39][20,39]. Furthermore, the loss of core PCP proteins abrogates the orientation of cilia, suggesting that the asymmetric distribution of core PCP proteins provides a cue to orient cilia. In contrast to Drosophila genes encoding core PCP proteins, those in vertebrates are often duplicated and might have divergent functions (Figure 2A”). For example, there are three homologs of Drosophila Flamingo, CELSR1, 2, and 3 in mice, and these have different expression patterns and show different phenotypes when their functions are lost ^{[20][40][41][42]}[20,40,41,42].

2.1. Multicellular and Tissue-Level Coordination of Cilia Orientation

An important role of core PCP proteins in PCP establishment is their non-cell-autonomous effect on the orientation of adjacent cells [23]. CELSRs are atypical cadherins and their homophilic interactions through their extracellular domains enable the intercellular coupling of FZD- and VANGL-complexes (Figure 2A,A’) [23]. In Xenopus skin, the transplantation of VANGL2-overexpressing tissues showed non-cell autonomous effects on adjacent wild type cells. In these wild type cells, cilia pointed away from the transplant. VANGL2-knocked down transplants showed opposite effects to the overexpressing transplants. In other words, cilia always pointed to cells with lower VANGL2-level [43]. In the oviduct of CELSR1 knockout mice, cilia orientation is still coordinated in each cell, but the mean angle of cilia orientations in each cell varies among adjacent cells [39]. These observations suggest that core PCP proteins are required for the intercellular coordination of cilia orientation [41]. To align the orientation of cells along the tissue axis, factors that transmit information regarding tissue orientation are necessary. Such factors are referred to as global factors, which include a gradient of extracellular concentration of WNT molecules ^[44][45][46][44,45,46], differences in the level of expression of atypical cadherins Dachsous/Fat and their modulator Four-jointed ^{[23][25][47][48][49][50]}[23,25,47,48,49,50], forces exerted on epithelial tissues ^{[51][52][53][54]}[51,52,53,54], and fluid flow ^[37][55][37,55]. Those global cues somehow control the localization of core PCP proteins at cell boundaries. For example, in Xenopus skin, core PCP proteins are stabilized at cell boundaries that lie perpendicular to the direction of gastrulation. When mechanical strain was artificially applied to Xenopus skin, localization of core PCP proteins was stabilized at cell boundaries, suggesting that the mechanical strain generated by gastrulation acts as a global cue [54]. However, mechanisms by which global cues control the localization of core PCP proteins, and what acts as a global cue in each organ, are still largely unknown.

2.2. How Do Core PCP Proteins Orient Cilia?

Various forms of evidence suggest that the distribution of core PCP proteins at the cell boundary provides a directional cue for orienting cilia. If so, how do core PCP proteins control cilia that emerge from distant locations? Recent studies suggest that core PCP proteins orient cilia via microtubules ^{[38][56][57][58]}[38,56,57,58]. In ependymal cells of the ventricle, a molecular motor dynein is localized at the cell cortex where the FZD-complex is enriched. Dishevelled-associating protein DAPLE is required for the localization of dynein at the cell cortex, and the loss of DAPLE and the inhibition of the activity of dynein both abrogate the orientation of cilia ^[56][59][56,59]. It has been proposed that dynein at the cell cortex pulls microtubules that connect the cell cortex and basal bodies, and this pulling force might orient cilia to FZD-enriched cell boundaries (Figure 2C) [56]. DAPLE is also required for the establishment of rotational polarity in the trachea, although it is unclear whether the role of DAPLE is the same as in ependymal cells. In the trachea, DAPLE binds to FZD6, and bundles and stabilizes nearby microtubules, and this concentrates microtubules around the FZD6-enriched cell cortex [58]. A theoretical analysis incorporating a hydrodynamic interaction between cilia and microtubules suggests that such an asymmetric concentration of microtubules is sufficient to orient cilia [57]. A similar asymmetric concentration of microtubules was reported in the mouse oviduct [39]. In a CELSR1 mutant oviduct, microtubules were still concentrated in more than 75% of multi-ciliated cells, while the orientation of the concentration was not aligned along the tissue axis. Importantly, the orientation of the concentration was consistent with that of cilia in each CELSR1 mutant cell [39]. Therefore, CELSR1 control the orientation of the concentration of microtubules, thus aligning cilia along the body axis. These results suggest that core PCP proteins provide directional information to cilia via microtubules that is sensed by basal bodies. Interestingly, in the Xenopus skin, an effective stroke pointed to the direction of VANGL-complex-enriched cell boundaries [60]. This relationship is reversed in the mouse oviduct, trachea, and ventricle, where the direction of recovery stroke and that of VANGL complex-enriched cell boundaries are consistent ^[37][38][42][37,38,42]. Mechanisms of how core PCP proteins control cilia orientation might be different among tissues and animals.

2.3. Variable Roles of Members of Core PCP Proteins in Multi-Ciliated Cells

A simplified view of how core PCP proteins orient cilia is as follows: (1) global cues orient the cell boundary-localization of core PCP proteins along the tissue axis and (2) core PCP proteins orient cilia toward core PCP proteins-enriched cell boundaries via microtubules. However, complexities reside in this core PCP proteins-dependent mechanism. Genetic analyses suggest that each member of core PCP proteins plays different roles in multi-ciliated cells. In the brain ventricle, intercellular coordination of cilia orientation require CELSR1, while rotational polarity depends on CELSR3 and VANGL2 [41]. In addition to cell boundaries, core PCP proteins are also detected at cilia. DVLs are localized at the base of cilia in Xenopus skin, mouse trachea and ventricle ^[37][38][61][37,38,61], and VANGL2 is localized along the cilia in mouse ventricle [37]. DVL1, DVL2, and DVL3 seem to show different subcellar localization in a tissue-dependent manner. Whereas DVL1 and DVL3 are localized at cell boundaries, DVL2 is localized only at the base of cilia in mouse trachea [38]. In the mouse ventricle, DVL1 and DVL2 are detected at the patch of basal bodies ^[37][62][63][37,62,63]. In Xenopus skin, knockdown of DVLs disrupted the apical migration of basal bodies, and impacted ciliogenesis. Furthermore, when a deletion form of DVL, Xdd1, was expressed in multi-ciliated cells, ciliogenesis was weakly affected, but the orientation of cilia was no longer aligned in each cell. In addition to Xdd1, misexpression of a dominant negative form of RhoA severely misoriented cilia. Since rGBD, which binds to active RhoA, was concentrated in foci at the apical surface of multi-ciliated cells and those foci were lost when DVLs became depleted, DVLs might control cilia orientation via the activation of RhoA at basal bodies (Figure 2C) [61]. Furthermore, functions of DVL in cilia might be regulated post-transcriptionally. PTEN dephosphorylates serine 143 of DVL2, and the loss of PTEN affects ciliogenesis and/or the polarity of cilia in Xenopus skin, and mouse trachea and ventricle [64]. In addition to DVLs, CELSRs also regulate ciliogenesis. In ventricles lacking both CELSR2 and CELSR3, basal bodies do not migrate to the apical surface of cells during the differentiation of multi-ciliated cells [36]. These findings indicate that core PCP proteins function at two different locations, at cilia and at cell boundaries.