Drosophila Crumbs (Crb) is required for apical-basal polarity and is an apical determinant in embryonic epithelia. Here, we describe properties of Crb that control the position and integrity of the photoreceptor adherens junction and photosensitive organ, or rhabdomere. In contrast to normal photoreceptor adherens junctions and rhabdomeres, which span the depth of the retina, adherens junctions and rhabdomeres of Crb-deficient photoreceptors initially accumulate at the top of the retina and fail to maintain their integrity as they stretch to the retinal floor. We show that Crb controls localization of the adherens junction through its intracellular domain containing a putative binding site for a protein 4.1 superfamily protein (FERM). Although loss of Crb or overexpression of the FERM binding domain causes mislocalization of adherens junctions, they do not result in a significant loss of photoreceptor polarity. Mutations in CRB1, a human homologue of crb, are associated with photoreceptor degeneration in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA). The intracellular domain of CRB1 behaves similarly to its Drosophila counterpart when overexpressed in the fly eye. Our studies may provide clues for mechanisms of photoreceptor degeneration in RP12 and LCA.

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The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Dpatj (Drosophila homolog of mammalian Patj) is a multi-PDZ domain protein that localizes to the apical cell membrane and forms a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (Sdt). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the dpatj gene.

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.

BackgroundMutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.Methodology/Principal FindingsThe spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin's gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.Conclusions/SignificanceThese results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.

Spectrins are major proteins in the cytoskeletal network of most cells. In Drosophila, beta(Heavy)-Spectrin encoded by the karst gene functions together with Crb during photoreceptor morphogenesis. However, the roles of two other Spectrins (alpha- and beta-Spectrins) in developing photoreceptor cells have not been studied. Here, we analyzed the effects of spectrin mutations on developing eyes to determine their roles in photoreceptor morphogenesis. We found that the Spectrins are dispensable for retinal differentiation in eye imaginal discs during larval stage. However, photoreceptors deficient in alpha- or beta-Spectrin display dramatic apical membrane expansions including Crb and show morphogenesis defects during pupal eye development, suggesting that alpha- and beta-Spectrins are specifically required for photoreceptor polarity during pupal eye development. Karst localizes apically, whereas beta-Spectrin is preferentially distributed in the basolateral region. We show that overexpression of beta-Spectrin causes a strong shrinkage of apical membrane domains, and loss of beta-Spectrin causes an expansion of apical domains, implying an antagonistic relationship between beta-Spectrin and Karst. These results indicate that Spectrins are required for controlling photoreceptor morphogenesis through the modulations of cell membrane domains.

Summary: Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. Here, we provide evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single-or few-celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par-3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type-specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis. genesis 48:522-530, 2010. V V C 2010 Wiley-Liss, Inc.

BackgroundCrumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.Methodology/Principal FindingsBecause a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.Conclusions/SignificanceIn summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.