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Drosophila Rab23 Is Involved in the Regulation of the Number and Planar Polarization of the Adult Cuticular Hairs

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Drosophila Rab23 Is Involved in the Regulation of the Number and Planar Polarization of the Adult Cuticular Hairs Copyright Ó 2010 by the Genetics Society of America DOI: 10.1534/genetics.109.112060 Drosophila Rab23 Is Involved in the Regulation of the Number and Planar Polarization of the Adult Cuticular Hairs Csilla Pataki,*,1 Tama´s Matusek,*,1 E´ va Kurucz,* Istva´n Ando´,* Andreas Jenny† and Jo´zsef Miha´ly*,2 *Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged 6726, Hungary and †Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Manuscript received November 13, 2009 Accepted for publication January 27, 2010 ABSTRACT 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, we identified Rab23, encoding a putative vesicular trafficking protein. Mutations in the Drosophila Rab23 ortholog result in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. We show that Rab23 is required for hexagonal packing of the wing cells. We found that 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, our 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. HE formation of properly differentiated organs Mutations in PCP genes result in abnormal wing hair T often requires the planar coordination of cell polarity patterns and wing hair number (Gubb and polarization within tissues, a feature referred to as Garcia-Bellido 1982; Wong and Adler 1993). On the planar cell polarity (PCP) or tissue polarity. Although basis of their cellular phenotypes (i.e., prehair initiation planar polarity is evident in many vertebrate tissues site and number of hairs per cell), initial studies placed (such as fish scales, bird feathers, and cochlear epi- PCP genes into three groups: the first group (often thelium) and it has recently been shown that PCP called the core group) includes frizzled (fz), dishevelled regulation is highly conserved throughout the animal (dsh), starry night (stan) (also known as flamingo), Van kingdom (Strutt 2003; Fanto and McNeill 2004; Gogh (Vang) (also known as strabismus), prickle (pk), and Seifert and Mlodzik 2007; Simons and Mlodzik diego (dgo); the second group consists of inturned (in), 2008), such polarization patterns are best studied in the fuzzy (fy), and fritz (frtz) (referred to as planar polarity fruitfly, Drosophila melanogaster. PCP in flies is manifest in effectors or In group); whereas the third group includes the mirror-image arrangement of ommatidia in the eye, multiple wing hairs (mwh)(Wong and Adler 1993). in the adult cuticle, which is decorated with parallel Double mutant analysis demonstrated that these phe- arrays of hairs and sensory bristles, and in the wing, notypic groups also represent epistatic groups, and it which is covered by distally pointing hairs (or tri- was proposed that the PCP genes may act in a regulatory chomes) (Adler 2002). Wing hairs form during the hierarchy, where the core group is on the top, whereas pupal life when each cell produces a single microvillus- the In group and mwh are downstream components like prehair stiffened by actin and microtubules. In (Wong and Adler 1993). Subsequent work identified wild-type wing cells prehairs form at the distal vertex of several other PCP genes as well. Some of these have been the cells and extend distally as they grow (Wong and placed into the Fat/Dachsous group (Adler et al. 1998; Adler 1993). Strutt and Strutt 2002), while another group con- sists of cytoskeletal regulators, including Rho1 and Drok (Strutt et al. 1997; Winter et al. 2001; Adler 2002). Supporting information is available online at http://www.genetics.org/ Genetic analysis of these two groups has led to models in cgi/content/full/genetics.109.112060/DC1. which the Fat/Dachsous group acts upstream of the 1 These authors contributed equally to this work. core proteins (Yang et al. 2002; Ma et al. 2003), while 2Corresponding author: Institute of Genetics, Biological Research Center, trutt Hungarian Academy of Sciences, Temesva´ri krt. 62, Szeged H-6726, Rho1 and Drok act downstream of Fz (S et al. 1997; Hungary. E-mail: [email protected] Winter et al. 2001). Although the existence of a single, Genetics 184: 1051–1065 (April 2010) 1052 C. Pataki et al. linear PCP regulatory pathway is debated (Casal et al. (Emery et al. 2005). The Rab23T69A allele has been isolated 2006; Lawrence et al. 2007), it is clear that in the wing, during an F2 FRT/Flp (Xu and Rubin 1993) mosaic muta- PCP genes regulate (1) the number of prehairs, (2) the genesis screen. FRT82B chromosomes were mutagenized with ENU (1.6 mm), mutant clones were induced by Ubx-Flp, and place of prehair formation, and (3) wing hair orientation. PCP phenotypes were analyzed on the wing and notum. The While the molecular mechanism that restricts prehair Rab2351 excision allele was generated by remobilization of the formation to the distal vertex of the wing cells is elusive, P(RS5)5-SZ-3123 (DrosDel) P-element insertion line by using it has been well established that the core PCP pro- standard techniques. The Rab23 RNAi line has been provided teins adopt an asymmetrical subcellular localization by the VDRC RNAi Centre (IMP-IMBA, Vienna), whereas the sui xelrod w; UASp-YFP-Rab23 lines were generous gifts from M. Scott when prehairs form (U et al. 1999; A 2001; (Stanford University). Flip out clones of UASp-YFP-Rab23 were Shimada et al. 2001; Strutt 2001; Tree et al. 2002; generated using w, hsFLP; ActP-FRT-y1-FRT-Gal4, arm-lacZ with Bastock et al. 2003; Das et al. 2004), which appears to be a 2-hr heat shock at 37° during the early third instar larval critical for proper trichome placement. In addition, it stage. For overexpression studies we used Act5C-Gal4 or Sal- has recently been found that the In group of proteins Gal4. In line with FlyBase, we use starry night instead of flamingo, and Van Gogh instead of strabismus. and Mwh also display an asymmetrical pattern with Cuticle preparation: Abdominal cuticles and first thoracic accumulation at the proximal zone (Adler et al. 2004; legs were prepared according to a protocol by Duncan (1982). Strutt and Warrington 2008; Yan et al. 2008). These DNA techniques and tissue culture: DNA constructs for studies concluded that the core PCP proteins are transgenic flies and transfection experiments were created by symmetrically distributed until 24 hr after prepupa standard cloning techniques. Detailed cloning procedures can be obtained from the authors upon request. To create a Rab23 formation (APF), when they become differentially en- genomic rescue construct, a 12.3-kb XbaI restriction fragment riched until prehair formation begins at 30–32 hr APF. from BACR03P13 (BACPAC Resources) was cloned into a This transient asymmetric localization ends by 36 hr pCaSpeR4 vector. To clone the Rab2351 transcript, total RNA APF (Adler 2002; Strutt 2003; Miha´ly et al. 2005). It was isolated from homozygous mutant adults with Trizol has recently been shown that Fz and Stan containing reagent (Invitrogen), cDNA was synthesized with Revert Aid (Fermentas), and RT–PCR was carried out with primers vesicles are transported preferentially toward the distal himada specific to the first and third exon of Rab23, respectively. cell cortex in the period of 24–30 hr APF (S et al. Primers used were as follows: Rab23Ex3 59-CCCGGCCACAA 2006), and hence, polarized vesicular trafficking might CATCATTAG-39 and Rab23Ex1 59-TCAAATTTCGATCGCGA be an important determinant of PCP protein asymme- CGAG-39. Molecular cloning of the RT–PCR product revealed 51 try. Other recent studies, however, challenged the view that Rab23 encodes a hybrid transcript consisting of the first (noncoding) exon of Rab23, part of the first intron of Rab23, that PCP protein polarization is limited to 24–32 hr APF. followed by 443 bp from the 59 end of the P(RS5)5-Sz-3123 Instead, it has been suggested that at least a partial insertion precisely until the end of ORF0 (O’Hare and Rubin proximal–distal polarization is already evident at the 1983) that is fused to the third exon of Rab23 (Figure 1B). If end of larval life and during the prepupal stages (6 hr translation from this fusion transcript begins with the ATG of APF). Polarity is then largely lost at the beginning of the ORF0, the predicted fusion protein is entirely devoid of Rab23 pupal period, but becomes evident again in several sequences because ORF0 and the third exon of Rab23 are not lassen in the same phase. If a downstream ATG were used, M53 of hours until hair formation begins (C et al. 2005). Rab23 is the next starting point. However, translational Thus, molecular asymmetries are clearly revealed dur- initiation from M53 would result in a mutant Rab23 protein ing wing hair formation, yet the molecular mechanisms that is lacking 52 N-terminal amino acids, including 15 of the that contribute to the establishment of these asymmet- highly conserved and functionally essential part of the GTPase antos ebreda rical patterns are not well understood.
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