The planar coordination of cellular polarization is an important, yet not well-understood aspect of animal development. In a screen for genes regulating planar cell polarization in Drosophila, Rab23, encoding a putative vesicular trafficking protein, was identified. Mutations in the Drosophila Rab23 ortholog result in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. Rab23 is required for hexagonal packing of the wing cells. Rab23 is able to associate with the proximally accumulated Prickle protein, although Rab23 itself does not seem to display a polarized subcellular distribution in wing cells, and it appears to play a relatively subtle role in cortical polarization of the polarity proteins. The absence of Rab23 leads to increased actin accumulation in the subapical region of the pupal wing cells that fail to restrict prehair initiation to a single site. Rab23 acts as a dominant enhancer of the weak multiple hair phenotype exhibited by the core polarity mutations, whereas the Rab23 homozygous mutant phenotype is sensitive to the gene dose of the planar polarity effector genes. Together, these data suggest that Rab23 contributes to the mechanism that inhibits hair formation at positions outside of the distal vertex by activating the planar polarity effector system (Pataki, 2010).

The formation of properly differentiated organs often requires the planar coordination of cell polarization within tissues, a feature referred to as planar cell polarity (PCP) or tissue polarity. Although planar polarity is evident in many vertebrate tissues (such as fish scales, bird feathers, and cochlear epithelium) and it has recently been shown that PCP regulation is highly conserved throughout the animal kingdom, such polarization patterns are best studied in Drosophila melanogaster. PCP in flies is manifest in the mirror-image arrangement of ommatidia in the eye, in the adult cuticle, which is decorated with parallel arrays of hairs and sensory bristles, and in the wing, which is covered by distally pointing hairs (or trichomes). Wing hairs form during the pupal life when each cell produces a single microvillus-like prehair stiffened by actin and microtubules. In wild-type wing cells prehairs form at the distal vertex of the cells and extend distally as they grow (Pataki, 2010).

Mutations in PCP genes result in abnormal wing hair polarity patterns and wing hair number. On the basis of their cellular phenotypes (i.e., prehair initiation site and number of hairs per cell), initial studies placed PCP genes into three groups: the first group (often called the core group) includes frizzled (fz), dishevelled (dsh), starry night (stan) (also known as flamingo), Van Gogh (Vang) (also known as strabismus), prickle (pk), and diego (dgo); the second group consists of inturned (in), fuzzy (fy), and fritz (frtz) (referred to as planar polarity effectors or In group); whereas the third group includes multiple wing hairs (mwh). Double mutant analysis demonstrated that these phenotypic groups also represent epistatic groups, and it was proposed that the PCP genes may act in a regulatory hierarchy, where the core group is on the top, whereas the In group and mwh are downstream components. Subsequent work identified several other PCP genes as well. Some of these have been placed into the Fat/Dachsous group, while another group consists of cytoskeletal regulators, including Rho1 and Drok. Genetic analysis of these two groups has led to models in which the Fat/Dachsous group acts upstream of the core proteins, while Rho1 and Drok act downstream of Fz. Although the existence of a single, linear PCP regulatory pathway is debated, it is clear that in the wing, PCP genes regulate (1) the number of prehairs, (2) the place of prehair formation, and (3) wing hair orientation (Pataki, 2010).

While the molecular mechanism that restricts prehair formation to the distal vertex of the wing cells is elusive, it has been well established that the core PCP proteins adopt an asymmetrical subcellular localization when prehairs form, which appears to be critical for proper trichome placement. In addition, it has recently been found that the In group of proteins and Mwh also display an asymmetrical pattern with accumulation at the proximal zone. These studies concluded that the core PCP proteins are symmetrically distributed until 24 hr after prepupa formation (APF), when they become differentially enriched until prehair formation begins at 30-32 hr APF. This transient asymmetric localization ends by 36 hr APF. It has recently been shown that Fz and Stan containing vesicles are transported preferentially toward the distal cell cortex in the period of 24-30 hr APF, and hence, polarized vesicular trafficking might be an important determinant of PCP protein asymmetry. Other recent studies, however, challenged the view that PCP protein polarization is limited to 24-32 hr APF. Instead, it has been suggested that at least a partial proximal-distal polarization is already evident at the end of larval life and during the prepupal stages (6 hr APF). Polarity is then largely lost at the beginning of the pupal period, but becomes evident again in several hours until hair formation begins. Thus, molecular asymmetries are clearly revealed during wing hair formation, yet the molecular mechanisms that contribute to the establishment of these asymmetrical patterns are not well understood (Pataki, 2010).

In a large-scale mosaic type of mutagenesis screen, Drosophila Rab23, encoding a vesicle trafficking protein, was identified as a PCP gene involved in the regulation of trichome orientation and number in various adult cuticular structures, including the wing, abdomen, and leg. This study shows that Rab23 plays a modest role in cortical polarization of the core PCP proteins in the wing and that Rab23 associates with at least one core protein, Pk. Additionally, it was found that Rab23 contributes to the mechanism that restricts actin accumulation and thus, prehair initiation to a single site within each wing cell (Pataki, 2010).

Rab23 appears to regulate two main aspects of trichome development, hair orientation and hair number. In pupal wing cells, the absence of Rab23 leads to increased actin accumulation in the subapical region and the formation of multiple hairs. In addition, Rab23 mutations impair hexagonal packing of the wing cells, and to a lesser degree, affect cortical polarization of the PCP proteins. Although, Rab23 does not appear to exhibit a polarized distribution in wing cells, it was found that Rab23 associates with Pk, which normally accumulates in the proximal cortical domain (Pataki, 2010).

Careful comparison of the Rab23 mutant phenotype with that of the other PCP mutations reveals that the phenotypic effect of Rab23 differs from all of the known PCP genes. Most notably, Rab23 has a specific requirement in the development of one particular type of subcellular structure (i.e., the cuticular hair) in every body region examined. However, it does not appear to play any role in the planar orientation of multicellular units such as ommatidia in the eye or the sensory bristles of the adult epidermis. In contrast to this, other PCP genes typically exhibit a tissue specific, but not structure specific, requirement, or, such as mutations of the core group, affect the polarization of every tissue and structure, regardless of whether they are hairs, bristles, or unit eyes. Focusing on the wing, loss of Rab23 results in weak trichome orientation defects and a relatively strong multiple hair phenotype (mostly double hairs). This is clearly different from the core PCP phenotypes (strong hair orientation defects and few multiple hairs), or the phenotypes of the In group and mwh (strong orientation defects and multiple hairs in almost every cell). As compared to Rho1 and Drok, Rab23 displays a similar adult wing hair phenotype in mutant clones with respect to multiple hairs, while the orientation defects are less clear in Rho1 and Drok mutants than in Rab23. Moreover, a significant difference exists at the molecular level, because, unlike Rab23, Rho1 and Drok do not play a role in cortical polarization of the core PCP proteins. Given that Rab23 alleles genetically behave as strong LOF or null alleles, Rab23 identifies a unique class of PCP genes dedicated to the regulation of trichome planar polarization (Pataki, 2010).

Although some recent data suggested that the establishment of properly polarized cortical domains is not an absolute requirement for correct trichome polarity in the wing, asymmetric accumulation of the PCP proteins is thought to serve as a critical cue for cell polarization. Thus, the Rab23-induced weak alterations in wing hair polarity are best explained by the similarly modest effect on PCP protein asymmetries. Because Rab23 is able to associate with Pk, it follows that Rab23 is likely to play a role in the proximal accumulation of Pk. Given that the Rab family of proteins is known to control membrane trafficking, the results provide further support for models suggesting that polarized membrane transport is an important mechanism for the asymmetric accumulation of the PCP proteins. Although Rab23 showed a specific interaction with Pk, technical limitations might have prevented the detection of interactions with other core PCP proteins, and hence it is possible that the mechanism whereby Rab23 contributes to cortical polarization is not limited to Pk regulation. One additional candidate is the transmembrane protein Vang that partly colocalizes with Rab23 in S2 cells and has been shown to bind Pk. Thus, through binding to Pk, Rab23 might affect Vang localization or signaling capacity. Irrespective of whether Rab23 directly affects the localization of only one or more PCP proteins, in the wing Rab23 has a relatively modest effect on protein localization, and, as a consequence, on hair orientation, indicating that Rab23 has a minor or largely redundant role in this tissue. Interestingly, however, Rab23 induces much stronger trichome orientation defects on the abdominal cuticle. Although it is not proven formally, genetic analysis suggests that asymmetric PCP protein accumulation (or at least polarized activation) is likely to occur in the abdominal histoblast cells as well. Hence, with respect to protein polarization Rab23 may act in a tissue-specific manner playing a largely dispensable role in the wing, but having a critical role in the abdominal epidermis (Pataki, 2010).

Correct trichome placement at a single distally located site is clearly a crucial step in planar polarization of the wing cells. Current models suggest that prehair initiation is controlled by an inhibitory cue localized proximally in a Vang-dependent manner, and by a Fz-dependent cue that positively regulates hair formation at the distal vertex (Strutt, 2008). Whereas it is not clear how the distal cues work, with regard to the proximal cues it is known that Vang and Pk colocalize with the effector proteins In, Fy and Frtz that control the localization and activity of Mwh, which is thought to regulate prehair initiation directly by interfering with actin bundling in the subapical region of cells (Strutt, 2008; Yan, 2008). This study found that Rab23 severely impairs trichome placement in the wing leading to the formation of multiple hairs, which indicates a role in the repression of ectopic hair initiation. Where does Rab23 fit into the regulatory hierarchy of trichome placement? Double mutant analysis suggests that Rab23 is upstream of the In group and mwh, and acts at the same level as the core PCP genes. The synergistic genetic interaction between Rab23 and the core PCP mutations indicates that they function in parallel pathways during the restriction of prehair initiation. Remarkably, the pk^pk; Rab23 double mutants exhibit an almost identical phenotype to mutations of the In group, suggesting that, unless the existence of an In independent restriction system is assumed, Pk and Rab23 together are both necessary and sufficient to fully activate the In complex. In pk single mutants the proximal accumulation of In is severely impaired, yet multiple hairs rarely develop, indicating that proper In localization plays only a minor role in the restriction mechanism. Conversely, in Rab23 single mutants In localization is weakly affected, but multiple hairs often form, suggesting that the major function of Rab23 is related to In activation. Thus, it appears that the proximally restricted activation of In on the one hand is ensured by Pk, that mainly plays a role in proper In localization, and on the other hand by Rab23, that seems to be required for In activation. At present, the molecular function of the In system is unknown, and it is therefore also unclear how Rab23 might contribute to the activation of the In complex. Nevertheless, because Rab23 has a weaker multiple hair phenotype than in, but the pk^pk; Rab23 double mutant is nearly as strong as in, it is conceivable that In activation is not exclusively Rab23 dependent but, beyond a role in protein localization, Pk has a partial requirement as well (Pataki, 2010).

The regulation of cellular packing is an interesting, yet only lately appreciated aspect of wing development. It has been reported that the wing epithelium is irregularly packed throughout larval and prepupal stages, but shortly before hair formation it becomes a quasihexagonal array of cells. Hexagonal repacking depends on the activity of the core PCP proteins. However, defects in packing geometry do not appear to directly perturb hair polarity in core PCP mutant wing cells. The possible exception to this rule is pk that exhibits very strong hair orientation defects and induces the strongest packing defects within the core PCP group. Additionally, another study revealed that irregularities in cell geometry are associated with polarity defects in the case of fat mutant clones. Thus, cell geometry is not the direct determinant of cell polarity, but in some instances cell packing seems to impact on PCP signaling and hair orientation. This study has shown that in the wing Rab23 is predominantly involved in the regulation of wing hair number, and it is also required for hexagonal packing of the wing epithelium. Do these packing defects correlate with the severity of the multiple hair phenotype? The data argue against this idea for the case of Rab23, and also for the cases of other strong multiple hair mutants, such as in, frtz, and mwh. Therefore, cell shape has no direct effect on the regulation of the number of prehair initiation sites, and Rab23 appears to regulate hexagonal packing and hair number independently (Pataki, 2010).

As Rab23 and Pk are both required for cellular packing, and Rab23 associates with Pk, it is possible that they cooperate during the regulation of packing. This is in agreement with the observation that pk^pk; Rab23 double mutant wings do not show stronger packing defects than a pk^pk single mutant. However, other interpretations are also possible, hence further investigations will be required to understand how Rab23 and Pk regulates cellular packing and to clarify the impact of packing geometry on PCP establishment in the wing (Pataki, 2010).

Unlike the vertebrate orthologs, Drosophila Rab23 is not an essential gene and does not appear to regulate Hedgehog signaling. Given that Rab GTPases are thought to regulate vesicular transport and that mouse Rab23 localizes to endosomes (Evans, 2003), it was expected that Rab23 regulates the trafficking of vesicle-associated Hedgehog signaling components. However, in the mammalian systems no clear link between endocytosis, Rab23, and the subcellular localization of Hedgehog signaling elements has been identified (Evans, 2003; Eggenschwiler, 2006; Wang, 2006). The finding that Rab23 associates with Pk suggests that Rab23 might be directly involved in the regulation of Pk trafficking, and therefore Pk could be the first known direct target of Rab23. Interestingly, there is a significant overlap reported in the embryonic expression domains of the vertebrate Pk and Rab23 genes in the region of the dorsal neural ectoderm, the somites, and the limb buds (Eggenschwiler, 2001; Wallingford, 2002; Takeuchi, 2003; Veeman, 2003; Li, 2007; Cooper, 2008). Moreover, it is also known that blocking of Rab23 or Pk function in vertebrate embryos can both lead to a spina bifida phenotype (Eggenschwiler, 2001; Wallingford, 2002; Takeuchi, 2003; Li, 2007). These observations raise the possibility that, unlike the Rab23 involvement in Hedgehog signaling, the Rab23-Pk regulatory connection is evolutionarily conserved (Pataki, 2010).