Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans

  • Fabio Demontis,

    Current address: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America

    Affiliation Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

  • Christian Dahmann

    dahmann@mpi-cbg.de

    Affiliation Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans

  • Fabio Demontis, 
  • Christian Dahmann
PLOS
x

Abstract

Background

The Usher syndrome (USH) is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively.

Principal Findings

Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche.

Conclusions

Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease.

Introduction

The analysis of orthologs of human disease genes in model organisms has revealed important insights into developmental mechanisms. One outstanding example is the contribution that the analysis of Usher syndrome-related genes has made to our understanding of hair cell development and auditory perception [1]. Usher syndrome is the most common hereditary disease associated with deafness and blindness in humans [2], [3], [4]. Three clinical subtypes, USH1, USH2, and USH3, have been defined according to the severity of the hearing impairment, the absence or presence of vestibular dysfunctions, and the age of onset of retinitis pigmentosa, which leads to blindness. USH1 is the most severe subtype and USH1 patients suffer from severe hearing impairment, balance defects, and prepubertal-onset of retinitis pigmentosa. Five USH1 genes have been identified: USH1B encodes an unconventional motor protein, myosin VIIa [5]; USH1C encodes a PDZ-domain-containing scaffolding protein, harmonin [5]; USH1D and USH1F encode two members of the cadherin family of Ca2+-dependent cell adhesion molecules, cadherin 23 and protocadherin 15, respectively [6], [7], [8], [9]; USH1G encodes a putative scaffolding protein, sans, containing three ankyrin repeats and a sterile alpha motif (SAM) domain [10].

Hair cells of the mammalian inner ear display bundles of actin-filled, microvilli-derived projections of the apical membrane, known as stereocilia, that act as mechanosensitive devices important for the detection of sound [1]. Stereocilia develop from microvilli through the lateral addition of actin filaments to the microvillar actin bundle and the subsequent elongation of the filaments within the actin bundle [11]. The differential elongation of stereocilia leads to their characteristic staircase-like arrangement. Within each bundle, the stereocilia are connected by various links, including tip links connecting the tip of one stereocilium to the side of an adjacent stereocilium. Mutations in the five known USH1 genes lead to alterations in stereocilia length and orientation. Biochemical analysis further shows that the five USH1 proteins can interact with each other in vitro. Harmonin binds, through its PDZ domain, to any of the other four USH1 proteins [10], [12], [13], [14], [15] and myosin VIIa can interact with protocadherin 15 and sans [12], [16]. Localization studies have revealed that four of the five USH1 proteins are present on stereocilia tips: myosin VIIa, cadherin 23, protocadherin 15, and harmonin [13], [17], [18]. The in vitro binding and in situ co-localization of these four USH1 proteins suggest that they form functional complexes. Cadherin 23 and protocadherin 15, furthermore, have been recently shown to be part of the tip links that connect stereocilia, thereby providing cohesion between adjacent stereocilia [19], [20]. Sans, in contrast to the other identified USH1 proteins, localizes to vesicular structures beneath the apical plasma membrane of hair cells [12] and has not been reported to reside on stereocilia. Based on its localization and physical interaction with myosin VIIa and harmonin, it has been proposed that sans may control the trafficking of USH1 proteins to stereocilia [12].

Animal models have been important in further understanding the etiology of Usher syndrome. Mice mutant for myosin VIIa (shaker-1), harmonin (deaf circler), cadherin 23 (waltzer), protocadherin 15 (Ames waltzer), or sans (Jackson shaker) display severe hearing impairment, vestibular dysfunction, and disorganized stereocilia in the inner ear [7], [18], [21], [22], [23], [24], [25]. Localization studies in mutant mice have shown that both myosin VIIa and sans are required to direct harmonin to the stereocilia tips [18]. Mutations in myosin VIIa, cadherin 23, and protocadherin 15a are also associated with disorganized hair bundles in zebrafish [26], [27], [28]. In Drosophila melanogaster, orthologs of USH1 genes have been identified. Mutations in crinkled, the fly ortholog of myosin VIIa [29], result in disorganization of Johnston's organ, the flies' auditory system, and in deafness [30], [31]. Moreover, Cad99C, the ortholog of protocadherin 15, localizes to microvilli of the fly follicular epithelium, and mutations in this cadherin result in the misorientation of microvilli [32], [33]. These findings indicate an evolutionary conserved function for this class of cadherins in organizing actin-containing cellular protrusions.

Here, in order to further understand the role of sans during development and disease, we identified and characterized a Drosophila melanogaster ortholog of this gene. We show that Drosophila Sans is expressed in tubular organs in the embryo, lens-secreting cone cells in the adult eye, and microvilli-displaying follicle cells during oogenesis. Similar to its vertebrate ortholog, Sans protein localizes to punctate structures we identified as early endosomes associated with the syntaxin Avalanche.

Results

The Drosophila genome encodes an ortholog of the human Usher syndrome type 1G protein sans

To identify the Drosophila ortholog of the human sans protein, we used the human sans protein sequence in a BLASTp search against the Drosophila melanogaster genome. The product encoded by gene CG13320 was most similar to human sans. Back-Blast using CG13320 protein as a query sequence retrieved sans as the most related protein in vertebrates. Overall, human sans and Drosophila CG13320 are 32% identical (168/524 identical amino acid residues). A higher sequence identity was detected in the N-terminus (53%; 15/28), the ankyrin repeats (60%, 53/88), and the SAM domain (39% (26/66). Human sans and Drosophila CG13320 share a conserved C-terminal PDZ-domain binding site (data not shown) and display a similar domain organization including three ankyrin repeats and one SAM domain (Fig. 1). Moreover, in a phylogenetic tree, CG13320 clustered in the same clade as vertebrate sans and harp (Fig. 1). Harp is an ankyrin and SAM-domain containing protein related to sans. We conclude that CG13320 is the Drosophila ortholog of human sans and refer to it as Sans (see also FlyBase at www.flybase.org).

thumbnail
Figure 1. Identification of a Drosophila melanogaster ortholog of human Usher syndrome type 1G protein sans.

Phylogenetic tree. Drosophila melanogaster Sans is closely related to vertebrate sans/Harp proteins. Harp/sans proteins contain three ankyrin repeats (blue rectangles) and one SAM domain (green triangle). Hs: Homo sapiens; Ag: Anopheles gambiae; Dm Drosophila melanogaster; Dr: Danio rerio; Mm: Mus musculus.

https://doi.org/10.1371/journal.pone.0004753.g001

Sans is expressed in tubular organs of the embryo and in ovarian follicle cells

To further characterize Drosophila Sans, we analyzed its expression pattern in embryonic, larval, and adult tissues by RNA in situ hybridization using a Sans-specific RNA probe. In the embryo, Sans mRNA was first detectable at stage 11 in the hindgut (Fig. 2A). In later stages, Sans mRNA was detected in the foregut, the hindgut, the tracheal placodes, the tracheal primary branches, and the six mechanosensory Keilin's organs (Fig. 2B–E′). During larval stages, Sans was expressed ubiquitously in eye-antennal, wing, leg, and haltere imaginal discs (data not shown). The adult Drosophila ovary is composed of chains of egg chambers proceeding through 14 stages from the germarium to the oviduct [34]. Each egg chamber consists of 16 germline cells, one oocyte and 15 nurse cells, encapsulated by a monolayer of somatic, epithelial follicle cells. Sans expression was confined to follicle cells contacting the oocyte in stage 9–10A egg chambers (Fig. 2F,H), a time when follicle cell microvilli are remodeled and elongate. Sans RNA localized to the apical side of follicle cells (Fig. 2F). No staining was detected using a sense RNA probe to Sans (Fig. 2G), indicating that the staining was specific.

thumbnail
Figure 2. Sans is expressed in tubular organs of the embryo and in ovarian follicle cells.

A–E′: Wild-type embryos of the indicated stages hybridized with a Sans-specific antisense RNA probe. A,B,C,D,E: Dorsal views. A′,B′,C′,D′: Lateral views. E′ is a magnification of E showing the Keilin's organs (arrowheads). F–G: Wild-type ovaries of the indicated stages hybridized with (F,H) a Sans-specific antisense RNA probe or (G) a sense RNA probe. Sans is expressed in follicle cells surrounding the oocyte in stage 10 egg chambers. Anterior is to the left.

https://doi.org/10.1371/journal.pone.0004753.g002

Sans is dispensable for fly development and fertility

To functionally analyze Sans, we generated four different mutant alleles of Sans by imprecise excision of EP-element GE10371 inserted in the first intron of the Sans gene (Fig. 3A,B). All four mutations deleted parts of the predicted coding sequence for Sans, but did not obviously alter neighboring genes. These mutations, however, affect the putative gene CG30487, whose predicted coding sequence lies on the complementary DNA strand within an intron of Sans. Analysis by in situ hybridization with a Sans specific RNA probe showed that Sans transcript could no longer be detected in Sans245/Sans245 mutant egg chambers (Fig. 3C,D), suggesting that expression of Sans was impaired from this mutant allele. To test this further, we performed Western-blot analysis of control and Sans mutant ovaries using a polyclonal anti-Sans antiserum raised against the full-length protein (see Materials and Methods). A protein of approximately 75 kDa was detected in wild-type or control heterozygous mutant ovaries (Fig. 3E). Proteins of approximately 50 kDa, possibly corresponding to truncate forms of Sans, were detected in Sans38/Sans38 mutant flies, suggesting that this allele is hypomorphic (Fig. 3E). However, no Sans protein was detected in Sans63/Sans63, Sans245/Sans245, and Sans254/Sans254 homozygous mutant flies (Fig. 3E), suggesting that these alleles are amorphic. Flies homozygous mutant for any of the four Sans alleles were adult viable, male and female fertile, and had no gross morphological defects (data not shown), indicating that Sans is not essential for fly development and fertility.

thumbnail
Figure 3. Genomic organization of Sans and characterization of Sans mutant alleles.

A: Genomic organization in the region of Sans and extent of the deletions in Sans mutant alleles. The position of the EP-element GE10371 which was used to generate the Sans mutants is indicated. B: Exon-intron structure of Sans. The scale and genomic position is the same as for the extent of the deletions shown in (A). The two Sans transcripts differ by an addition of seven nucleotides to the 5′ end of transcript A. C,D: Stage 10 (C) wild-type and (D) Sans245/Sans245 mutant egg chambers hybridized with a Sans-specific RNA probe. The hybridization signal is undetectable in the mutant. E: Western blot analysis of ovaries with an anti-Sans antiserum. Genotypes of the flies are indicated. The anti-Sans antiserum detects a protein of ∼75 kDa (arrow), the expected molecular weight for Sans, in extracts of wild-type (lane 1) and heterozygous (lanes 3,5,7), but not homozygous (lanes 2,4,6,8) Sans mutant ovaries. Subsequent blotting with an anti α-Tubulin antibody (lower panel) shows the loading of similar amounts of protein in each lane. Molecular weights are indicated to the left (in kDa).

https://doi.org/10.1371/journal.pone.0004753.g003

Sans, Cad99C, and Crinkled/Myosin VIIa localize to cone cells in the adult eye

In the vertebrate eye, a number of USH1 proteins have been localized to the ciliary and periciliary regions of photoreceptor cells (reviewed in [35]). In addition, myosin VIIa is expressed in the retina pigmented epithelium [36], a multifunctional epithelium placed between the photoreceptors and the vascular meshwork of the choroid layer. To determine the localization of the three USH1 orthologs Sans, Cad99C, and Crinkled/Myosin VIIa in the Drosophila eye, we stained adult eyes with antisera specific to these proteins. The adult compound eye is a regular hexagonal array of approximately 750 ommatidia. Each ommatidium consists of 8 photoreceptors, 4 cone cells, and pigment cells (Fig. 4A). The cone cells overlie the photoreceptors and secrete lens material from their apical side. Photoreceptors display rod-like stacks of photosensitive microvilli at their apical surfaces, called rhabdomeres. The overall morphology of rhabdomeres, as visualized by staining for F-actin, was indistinguishable between control and Sans63/Sans63 mutant flies (Fig. 4B,C). Sans immunoreactivity was strongly detected in cone cells (Fig. 4D). No immunoreactivity was detected in the adult eyes of Sans63/Sans63 mutant flies (Fig. 4E), indicating that the observed staining was specific to Sans. In addition, we detected Cad99C immunoreactivity at what appeared to be the apical side of cone cells in control, but not in Cad99C57A/Cad99C57A mutant flies (Fig. 4F,G). Finally, Crinkled/Myosin VIIa immunoreactivity was detected in cone cells in control, but not in ck13/ck13 mutant flies (Fig. 4H and data not shown). We conclude that Sans, Cad99C, and Crinkled/Myosin VIIa localize to cone cells in adult eyes.

thumbnail
Figure 4. Localization of Sans, Cad99C, and Crinkled proteins in adult retinae.

A: A scheme of a longitudinal section of a Drosophila adult ommatidium. B–C: Longitudinal optical sections of adult retinae from (B) wild-type and (C) Sans63/Sans63 mutant animals stained for Rhodamine-Phalloidin (red) to detect Filamentous (F-) actin. D–E: Longitudinal optical sections of adult retinae from (D) wild-type and (E) Sans63/Sans63 mutant animals stained for Sans (green) and F-actin (red). F–G: Longitudinal optical sections of adult retinae from (F) wild-type and (G) Cad99C57A/Cad99C57A mutant animals stained for Cad99C (green) and E-cadherin (red). E-cadherin labels the adherens junctions between photoreceptor cells. H: Longitudinal optical sections of wild-type adult retinae stained for Crinkled (green) and Cad99C (red). Sans, Cad99C, and Crinkled localize to cone cells. Scale bars: 10 µm.

https://doi.org/10.1371/journal.pone.0004753.g004

Sans, Cad99C, and Crinkled/Myosin VIIa localize to follicle cells

Cad99C has previously been shown to localize to microvilli of follicle cells in stage 10 egg chambers [32], [33]. We next determined the expression and localization of Crinkled/Myosin VIIa and Sans in ovaries. crinkled RNA was detected in nurse cells and follicle cells of stage 9–10 egg chambers (Fig. 5A). In stage 11, crinkled RNA was confined to the nurse cells (Fig. 5B). No staining was detected using a sense RNA probe to crinkled (Fig. 5C), indicating that the staining was specific. Crinkled/Myosin VIIa immunoreactivity was first detected during oogenesis at the apical side of follicle cells in stage 7 egg chambers (Fig. 5D,E). In stage 10 egg chambers, immunoreactivity was detected in the follicle cells surrounding the oocyte and in the germline (Fig. 5F), consistent with the expression of crinkled RNA. Similar to crinkled RNA, Crinkled/Myosin VIIa immunoreactivity was no longer detected in follicle cells of stage 11 egg chambers (data not shown). Higher magnification views of stage 10 egg chambers showed that Crinkled/Myosin VIIa immunoreactivity was present in punctate structures at the apical side of follicle cells and, in part, co-localized with Cad99C (Fig. 5H). Crinkled/Myosin VIIa immunoreactivity was not obviously altered in ovaries of Sans245/Sans245 mutant flies (Fig. 5G,I). Sans immunoreactivity was detected in the most anterior part of the germarium in what appeared to be the germline stem cells (Fig. 6A,B). Moreover, Sans immunoreactivity was observed in the follicular epithelium surrounding the oocyte of control stage 9–10A egg chambers (Fig. 6D,D′), consistent with the pattern of Sans mRNA expression. No immunoreactivity was detected in ovarioles of Sans245/Sans245 mutant flies (Fig. 6C, E,E′), indicating that the observed stainings were specific for Sans. We conclude that Sans, Cad99C, and Crinkled/Myosin VIIa localize to follicle cells.

thumbnail
Figure 5. Expression and localization of Crinkled/Myosin VIIa in ovaries.

A–C: Wild-type ovaries of the indicated stages hybridized with (A,B) a crinkled-specific antisense RNA probe or (C) a sense RNA probe. crinkled is expressed in follicle cells surrounding the oocyte in stage 10 egg chambers. D–G: Ovaries of (D–F) wild-type and (G) Sans245/Sans245 mutant flies stained for Crinkled/Myosin VIIa (green) and F-actin (red). In (F,G) stage 10 egg chambers are shown. H,I: High magnification views of follicle cells of stage 10 egg chambers of (H) wild-type and (I) Sans245/Sans245 mutant flies stained for Crinkled/Myosin VIIa (green), F-actin (red) and Cad99C (white). Crinkled/Myosin VIIa localizes to punctate structures at the apical side of follicle cells and, in part, to Cad99C-stained microvilli in wild-type and Sans245/Sans245 mutant flies. Scale bars: D–G: 100 µm; H,I: 20 µm.

https://doi.org/10.1371/journal.pone.0004753.g005

thumbnail
Figure 6. Localization of Sans protein in ovaries.

A–E: Ovaries of (A,B,D) wild-type and (C,E) Sans245/Sans245 mutant flies stained for Sans (green), α-Tubulin (blue), and F-actin (red). (A–C) show the germarium and (D,E) stage 10 egg chambers. In (D′,E′) only the Sans staining is shown. Sans is expressed in what appears to be the germline stem cells of the germarium (A,B) and in follicle cells contacting the oocyte (D). Sans staining is also detected at the cortex of the oocyte. Anterior is to the left. Scale bars: A: 50 µm; B,C: 10 µm; D,E: 100 µm.

https://doi.org/10.1371/journal.pone.0004753.g006

Sans co-localizes with the syntaxin Avalanche to early endocytic vesicles in follicle cells

Next we analyzed in more detail the subcellular localization of Sans in follicle cells. Higher magnification views revealed that Sans localized to punctate structures within the follicle cell cytoplasm (Fig. 7A). Similarly, a YFP-Sans fusion protein, expressed using the GAL4-UAS-system, localized to intracellular punctate structures (Fig. 7B). Sans, as well as the YFP-Sans fusion protein, were enriched beneath the apical membrane of follicle cells (Fig. 7C–E).

thumbnail
Figure 7. Sans co-localizes with the syntaxin Avalanche to early endocytic vesicles.

A: Clones of Sans245/Sans245 mutant follicle cells marked by the absence of nuclear GFP (green) of stage 10-egg chambers stained for F-actin (red) and Sans (blue). Sans localizes to intracellular punctate structures of follicle cells. B–D: Clones of follicle cells expressing YFP-Sans (Act5C>Gal4, UAS-YFP-Sans) labeled by YFP-Sans (green) and stained for F-actin (red). The white line in (D) delineates the clone border. YFP-Sans localizes to punctate structures enriched beneath the apical plasma membrane. Note that the YFP-Sans punctate structures are enlarged in comparison to the endogeneous Sans punctate structures. E,F: Wild-type stage 10-egg chambers stained for Avalanche (Avl, red) and Sans (green). Sans partially co-localizes with Avl in punctate structures. (A,F) show top views and (B–E) show cross-sectional views of the follicle epithelium (apical is to the top). Scale bars: A,C–F: 10 µm; B: 20 µm.

https://doi.org/10.1371/journal.pone.0004753.g007

The syntaxin Avalanche (Avl) has been previously shown to localize in follicle cells to punctate structures within the cytoplasm below the apical plasma membrane [37], similar to Sans. Avl is present in an early compartment of the endocytic pathway and is required for endocytosis of the Notch and Crumbs transmembrane proteins [37]. To test whether Sans and Avl localize to the same punctate structures, we stained follicle cells for Sans and Avl. Sans-labelled punctate structures frequently co-localized with Avl (Fig. 7E,F). Image analysis revealed that 78% (n = 319) of the Sans-labelled punctate structures were also co-labeled by the Avl antibody. We conclude that Sans, at least in part, localizes to Avl-associated early-endocytic vesicles.

Sans is apparently not required for the normal morphogenesis of follicle cell microvilli

Mutations in Cad99C, the ortholog of the human Usher syndrome type 1F protein protocadherin 15, result in an abnormal morphology of follicle cell microvilli [32], [33]. Similar to Cad99C, Sans is expressed in stage 9–10A follicle cells, prompting us to test whether Sans is also required for normal microvilli morphogenesis. To this end, we immunostained stage 10 egg chambers of control and Sans245/Sans245 mutant flies for F-actin, which identifies microvilli. As shown in Fig. 8 (A and C), the morphology of follicle cell microvilli, as revealed by this analysis, was indistinguishable between control and Sans245/Sans245 mutant flies. Moreover, follicle cell microvilli also appeared to be normal in mutants for crinkled (ck13)/myosin VIIA (Fig. 8B). Cad99C, a marker for microvilli [32], [33], appeared to localize normally on microvilli of ck13/ck13 mutant follicle cells, as analyzed by immunofluorescence (Fig. 8A,B). Cad99C also localized to microvilli in Sans245/Sans245 mutant follicle cells, even though Cad99C staining on microvilli appeared, in some instances, to be less pronounced compared to controls (Fig. 8C,D). The total amount of Cad99C in ovaries, as analyzed by Western blotting, was similar in control and Sans mutant flies (Fig. 8E). Thus, unlike Cad99C, Sans does not appear to play an essential role during microvilli morphogenesis in the follicle epithelium.

thumbnail
Figure 8. Follicle cell microvilli display an apparently normal morphology in Sans and ck mutant flies.

A–C: Cross-sectional views of (A) wild-type, (B) ck13/ck13 mutant, and (C) Sans245/Sans245 mutant follicle cells of stage 10-egg chambers stained for F-actin (red), Cad99C (green), and α-Tubulin (blue). At this level of resolution, microvilli appear to be normal in the mutants. D: Clones of Sans245/Sans245 mutant follicle cells marked by the absence of GFP (blue) of stage 10-egg chambers stained for F-actin (red) and Cad99C (green). A cross-sectional view is shown (apical is to the top). D′ displays a heat-map of the staining shown in D. Cad99C is reduced on microvilli of Sans245/Sans245 mutant follicle cells. E: Western blot analysis of ovaries with an anti-Cad99C antiserum. Genotypes of the flies are indicated. Similar amounts of Cad99C (arrow) are detected in extracts of wild-type (lane 1), heterozygous (lanes 3,5,7), and homozygous (lanes 2,4,6,8) Sans mutant ovaries. Subsequent blotting with an anti α-Tubulin antibody (lower panel) shows the loading of similar amounts of proteins in each lane. Molecular weights are indicated to the left (in kDa). Scale bars: A–C: 10 µm; D: 20 µm.

https://doi.org/10.1371/journal.pone.0004753.g008

Discussion

In this study, we have identified and characterized a Drosophila ortholog of the Usher syndrome protein sans. Drosophila Sans is expressed in tubular organs of the embryo and in cone cells in the adult eye. In the ovary, Sans is expressed in germline stem cells and in follicle cells during stages 9–10A. In follicle cells, Sans colocalizes with Avalanche, a syntaxin associated with early endosomes.

Orthologs of USH1 genes in Drosophila

The Drosophila melanogaster genome encodes genes orthologous to the five known USH1 genes: crinkled/myosin VIIa (USH1B) [29], CG5921 (USH1C) (data not shown), Cad88C (USH1D) [38], Cad99C (USH1F) [32], [33], and Sans (USH1G) (this study), providing an opportunity to study the cellular and developmental roles of these genes in this genetically tractable model organism. The analysis of crinkled has revealed a striking parallel in the role of this gene and USH1B for auditory perception. crinkled mutants fail to properly develop the flies auditory organs and they are deaf [30], [31]. Measurements of auditory mechanics and nerve responses indicate that Sans mutants do not have auditory defects (Martin Göpfert, personal communication). A further cell type in which orthologs of USH1 genes have been analyzed in Drosophila is the follicle epithelium [32], [33]. Like other epithelia, the follicle cells display microvilli at their apical surface. Follicle cell microvilli are dynamic in that they extend in length during stages 9–10A of oogenesis and that they disappear during the later stages. Interestingly, all five Usher syndrome type 1 orthologs, Cad99C, Cad88C, CG5921, crinkled, and Sans, are expressed in follicle cells when they display long microvilli [32], [33], [39] (this study). At least three Usher syndrome type 1 proteins share similar subcellular localizations in Drosophila follicle cells and vertebrate hair cells. Cad99C/protocadherin 15 and Crinkled/myosin VIIa localize to microvilli/stereocilia. Sans localizes to vesicles beneath the apical surface of cells. Moreover, in the adult Drosophila eye, Sans, Cad99C, and Crinkled/Myosin VIIa localize to a single cell type, the lens-secreting cone cell. Based on their localization, we speculate that Sans, Cad99C, and Crinkled/Myosin VIIa might be part of a protein network, similar to what has been proposed for their vertebrate orthologs [12], [16].

Sans and microvilli morphogenesis

Mutations in Cad99C result in misorientation of follicle cell microvilli [32], [33]. Normal microvilli orientation appears physiologically important, since misorientation of microvilli in Cad99C mutants correlates with an impairment of eggshell deposition necessary to protect the egg and developing embryo from its environment [32], [33]. In contrast to Cad99C mutants, follicle cell microvilli appear to be normal in both Sans and crinkled mutants, although we cannot exclude subtle defects. Sans mutant flies also deposit eggs surrounded by an apparently integer eggshell, as revealed by the exclusion of the dye neutral red (data not shown). In contrast to the vertebrate hair cells, microvilli morphogenesis in the follicle epithelium appears to be more robust towards single gene perturbations. We find that, although single mutants for Sans or crinkled are adult viable [29] (this study), animals double mutant for these genes do not develop to adulthood (data not shown), underscoring the developmental role of Usher syndrome orthologs in Drosophila. Future work will be needed to analyze microvillar morphogenesis in cells double mutant for Sans and crinkled and to determine the roles of the two thus far uncharacterized Usher type 1 orthologs Cad88C and CG5921 during Drosophila development.

A role for Sans in vesicle trafficking?

Based on the vesicle-like localization of sans in mammalian hair cells, it has been speculated that sans might be involved in vesicle trafficking and, in particular, the trafficking of cadherin 23 or protocadherin 15 to stereocilia [12]. However, the identity of the vesicle to which sans associated had not been revealed. We now show that, in Drosophila follicle cells, Sans to a large extent co-localizes with the syntaxin Avalanche to endocytic vesicles beneath the apical plasma membrane. These results are consistent with an evolutionary conserved role of Sans in vesicle trafficking. The decreased amount of Cad99C on microvilli, which we detected with low penetrance in Sans245/Sans245 mutant follicle cells is, furthermore, suggestive of a function of Sans in the trafficking of Cad99C. Our work provides a basis for the further analysis of Usher syndrome-related genes in Drosophila.

Materials and Methods

Bioinformatic analysis

BLAST searches [40] were performed against the non-redundant protein National Center for Biotechnology Information (NCBI) database. The Simple Modular Architecture Research Tool (SMART) [41] was used to predict the domain organization of sans-related molecules across evolution. Phylogenetic analysis was performed as previously described [42]. Accession numbers of the sequences used in this study are: Hs BAC85629, Hs CAH18298, Hs SANS (NP_775748), Mm SANS (NP_789817), Hs Harp (NP_665872), Mm Harp (NP_082361) [43], Dm AAQ21582 (DSANS), Ag XP_308746, Dr NP_001002188, Dr XP_696799.

Molecular cloning

To clone the CG13320/Sans coding sequence together with its 5′ and 3′ untranslated regions, we first isolated total RNA from third instar y w larvae using the RNeasy mini kit (Qiagen). cDNA was synthesized with the SMART RACE cDNA Amplification kit (Clontech) and used as template in a PCR reaction using the primers 5′-CCGGAATTCGCTTCCGGGTAACGGCTTTCGGAACG-3′ and 5′-CGGGGTACCGGAGTGATAATTGTTAGGCTTTAATTTAG-3′ (the underlined sequences are EcoRI and KpnI restriction sites used for cloning), corresponding to nucleotides 1–27 and 1833–1861 of the EST sequence LD20463. The sequencing of distinct PCR products revealed the presence of two different isoforms of CG13320 transcripts, one corresponding to the LD20463 sequence (+7nn isoform) and the other lacking 7 nucleotides in the 5′ untranslated region (nucleotides 163–169 of LD20463, −7nn isoform). The translation initiation site was identified at position 249 in the sequence NM_176160 (position 216 in the sequence LD20463) on the basis of the presence of a Kozak consensus sequence (cgaatATG), partially corresponding to the consensus sequence previously reported [44]. The translation initiation site was preceded by Stop codons in all three reading frames. The CG13320 gene encodes a protein of 516 amino acids that differs at positions 405 (T405S) from the sequence deposited for CG13320 (AAQ21582), probably due to polymorphisms between the Drosophila strains used for genome sequencing and the y w strain used in this study.

To generate the UAS-YFP-Sans transgene, the CG13320 coding sequence, together with the 3′ untranslated region, was amplified by PCR using the primers 5′-CCGGAATTCATGTCATCGGATCGG TTTCACAAAGC-3′ and 5′-CGGGGTACCGGAGTGATAATTGTTAGGCTTT AATTTAG-3′ (the underlined sequences are EcoRI and KpnI restriction sites used for cloning), corresponding to nucleotides 216–241 and 1833–1861 of LD20463. The resulting PCR product was cloned in the pUAST vector [45]. Enhanced yellow fluorescent protein (EYFP) was PCR amplified from pEYFP-N1 (Clontech), using the primers 5′-CCGGAATTCGTCGCCACCATGGTGAGCAA GG-3′ and 5′-CCGGAATTCCTTGTACAGCTCGTCCATGCC-3′ (the underlined sequences are EcoRI restriction sites used for cloning). The resulting PCR product was cloned 5′ to and in frame with the Sans coding sequence in the pUAST vector. The correct nucleotide sequence of the cloned PCR products were confirmed by sequencing prior to injection into y w embryos to obtain transgenic flies.

Fly stocks

Mutant alleles for CG13320 were generated by imprecise excision of EP-elements [46] using standard procedures. The starting EP-element line was GE10371 (GenExel, Inc.), in which the EP-element is oriented with the UAS sites facing away from the CG13320 coding sequence. The deficiency line used was Df(2R)CX1, wg12 b1 pr1/SM1 (Bloomington Drosophila stock center at Indiana University). The allele Sans38 contains a genomic deletion of 2135 bp, spanning from nucleotide 121224 to 123357 of the genomic sequence AE003820. The allele Sans63 contains a genomic deletion of 965 bp from nucleotide 122363 to nucleotide 123328. The allele Sans245 contains a genomic deletion of 1331 bp, spanning from nucleotide 122367 to nucleotide 123696. The allele Sans254 contains a genomic deletion of 1593 bp, spanning from nucleotide 121561 to nucleotide 123152. The four Sans alleles described here are homozygous viable, indicating that CG13320 (Sans) is not essential for viability. This conclusion is consistent with previous independently conducted genetic studies which did not uncover any vital gene between CG3845 (l(2)01424) and CG3886 (Psc) [47], [48], an interval including Sans. Additional fly stocks used were FRT42D Sans245 (this study), UAS-YFP-Sans (this study), Act5C>CD2>Gal4 [49], and ck13 [29]. Marked clones of mutant cells were generated by Flp-mediated mitotic recombination subjecting flies once to a 35°C heat-shock for 30 minutes (Act5C<CD2>Gal4) or three times to a 38°C heat shock for 30 minutes (FRT42D Sans245).

RNA in situ hybridization

RNA in situ hybridization was performed as described previously [33]. The EST LD20463 was used as a template for RNA probe preparation.

Western blotting

Western blotting was performed as described previously [33]. Primary antibodies used were rabbit anti-Sans (1∶1000; this study), rabbit anti-Cad99C, 1∶1000 [42] and mouse anti-α-Tubulin, 1∶1000 (Sigma T9026). Secondary antibodies used were HRP-conjugated goat anti-rabbit, 1∶5000 (Santa Cruz sc-2054) and HRP-conjugated goat anti-mouse, 1∶5000 (Pierce Biotechnology 31430).

Immunohistochemistry

Immunostaining of adult retinae was performed as described previously [50]. Ovaries were dissected and prepared for immunostaining using standard protocols. The anti-Sans antibody (AE5E) was generated by immunizing rabbits using a GST-Sans (full-length) fusion protein. The antiserum was immunopurified using a resin coupled to MBP-Sans. The anti-Sans antiserum was used at a 1∶100 dilution. Additional primary antibodies were monoclonal mouse anti-α-Tubulin, 1∶100 (Sigma T9026), monoclonal mouse anti-GFP, 1∶2000 (Clontech 8362-1), chicken anti-Avl, 1∶1000 [37], rat anti E-cadherin (DCAD2), 1∶20 [51], guinea pig anti-Crinkled, 1∶100 (Fig. 4) [29], mouse anti-MYO7A 138-1, 1∶100 (Fig. 5, Developmental Studies Hybridoma Bank), and rabbit anti-Cad99C, 1∶10.000 [42]. Secondary antibodies used were Alexa 488, Alexa 594 (Molecular Probes) or CY5 (Jackson ImmunoResearch) conjugated anti-mouse, anti-guinea pig, anti-rat, or anti-rabbit IgG, all diluted 1∶200. Rhodamine-Phalloidin, used at a 1∶200 dilution, was from Molecular Probes. Confocal images were recorded on a Zeiss LSM510 microscope.

Acknowledgments

We are grateful to M. Göpfert for performing the measurements of auditory mechanics and nerve responses. We thank GenExel and the Bloomington Drosophila Stock Center for fly stocks and D. Bilder and the Developmental Studies Hybridoma Bank for antibodies. We thank K. Schlichting for providing the data shown in Fig. 5D-I and E. Knust for critical comments on the manuscript.

Author Contributions

Conceived and designed the experiments: FD CD. Performed the experiments: FD. Analyzed the data: FD CD. Contributed reagents/materials/analysis tools: FD CD. Wrote the paper: FD CD.

References

  1. 1. Frolenkov GI, Belyantseva IA, Friedman TB, Griffith AJ (2004) Genetic insights into the morphogenesis of inner ear hair cells. Nat Rev Genet 5: 489–498.GI FrolenkovIA BelyantsevaTB FriedmanAJ Griffith2004Genetic insights into the morphogenesis of inner ear hair cells.Nat Rev Genet5489498
  2. 2. Ahmed ZM, Riazuddin S, Wilcox ER (2003) The molecular genetics of Usher syndrome. Clin Genet 63: 431–444.ZM AhmedS. RiazuddinER Wilcox2003The molecular genetics of Usher syndrome.Clin Genet63431444
  3. 3. El-Amraoui A, Petit C (2005) Usher I syndrome: unravelling the mechanisms that underlie the cohesion of the growing hair bundle in inner ear sensory cells. J Cell Sci 118: 4593–4603.A. El-AmraouiC. Petit2005Usher I syndrome: unravelling the mechanisms that underlie the cohesion of the growing hair bundle in inner ear sensory cells.J Cell Sci11845934603
  4. 4. Reiners J, Nagel-Wolfrum K, Jurgens K, Marker T, Wolfrum U (2006) Molecular basis of human Usher syndrome: deciphering the meshes of the Usher protein network provides insights into the pathomechanisms of the Usher disease. Exp Eye Res 83: 97–119.J. ReinersK. Nagel-WolfrumK. JurgensT. MarkerU. Wolfrum2006Molecular basis of human Usher syndrome: deciphering the meshes of the Usher protein network provides insights into the pathomechanisms of the Usher disease.Exp Eye Res8397119
  5. 5. Weil D, Blanchard S, Kaplan J, Guilford P, Gibson F, et al. (1995) Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 374: 60–61.D. WeilS. BlanchardJ. KaplanP. GuilfordF. Gibson1995Defective myosin VIIA gene responsible for Usher syndrome type 1B.Nature3746061
  6. 6. Ahmed ZM, Riazuddin S, Bernstein SL, Ahmed Z, Khan S, et al. (2001) Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F. Am J Hum Genet 69: 25–34.ZM AhmedS. RiazuddinSL BernsteinZ. AhmedS. Khan2001Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F.Am J Hum Genet692534
  7. 7. Alagramam KN, Murcia CL, Kwon HY, Pawlowski KS, Wright CG, et al. (2001) The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene. Nat Genet 27: 99–102.KN AlagramamCL MurciaHY KwonKS PawlowskiCG Wright2001The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene.Nat Genet2799102
  8. 8. Bolz H, von Brederlow B, Ramirez A, Bryda EC, Kutsche K, et al. (2001) Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D. Nat Genet 27: 108–112.H. BolzB. von BrederlowA. RamirezEC BrydaK. Kutsche2001Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.Nat Genet27108112
  9. 9. Bork JM, Peters LM, Riazuddin S, Bernstein SL, Ahmed ZM, et al. (2001) Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23. Am J Hum Genet 68: 26–37.JM BorkLM PetersS. RiazuddinSL BernsteinZM Ahmed2001Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23.Am J Hum Genet682637
  10. 10. Weil D, El-Amraoui A, Masmoudi S, Mustapha M, Kikkawa Y, et al. (2003) Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin. Hum Mol Genet 12: 463–471.D. WeilA. El-AmraouiS. MasmoudiM. MustaphaY. Kikkawa2003Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin.Hum Mol Genet12463471
  11. 11. DeRosier DJ, Tilney LG (2000) F-actin bundles are derivatives of microvilli: What does this tell us about how bundles might form? J Cell Biol 148: 1–6.DJ DeRosierLG Tilney2000F-actin bundles are derivatives of microvilli: What does this tell us about how bundles might form?J Cell Biol14816
  12. 12. Adato A, Michel V, Kikkawa Y, Reiners J, Alagramam KN, et al. (2005) Interactions in the network of Usher syndrome type 1 proteins. Hum Mol Genet 14: 347–356.A. AdatoV. MichelY. KikkawaJ. ReinersKN Alagramam2005Interactions in the network of Usher syndrome type 1 proteins.Hum Mol Genet14347356
  13. 13. Boeda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, et al. (2002) Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. Embo J 21: 6689–6699.B. BoedaA. El-AmraouiA. BahloulR. GoodyearL. Daviet2002Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.Embo J2166896699
  14. 14. Reiners J, Marker T, Jurgens K, Reidel B, Wolfrum U (2005) Photoreceptor expression of the Usher syndrome type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold protein harmonin (USH1C). Mol Vis 11: 347–355.J. ReinersT. MarkerK. JurgensB. ReidelU. Wolfrum2005Photoreceptor expression of the Usher syndrome type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold protein harmonin (USH1C).Mol Vis11347355
  15. 15. Siemens J, Kazmierczak P, Reynolds A, Sticker M, Littlewood-Evans A, et al. (2002) The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions. Proc Natl Acad Sci U S A 99: 14946–14951.J. SiemensP. KazmierczakA. ReynoldsM. StickerA. Littlewood-Evans2002The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions.Proc Natl Acad Sci U S A991494614951
  16. 16. Senften M, Schwander M, Kazmierczak P, Lillo C, Shin JB, et al. (2006) Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J Neurosci 26: 2060–2071.M. SenftenM. SchwanderP. KazmierczakC. LilloJB Shin2006Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells.J Neurosci2620602071
  17. 17. Ahmed ZM, Riazuddin S, Ahmad J, Bernstein SL, Guo Y, et al. (2003) PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23. Hum Mol Genet 12: 3215–3223.ZM AhmedS. RiazuddinJ. AhmadSL BernsteinY. Guo2003PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23.Hum Mol Genet1232153223
  18. 18. Lefevre G, Michel V, Weil D, Lepelletier L, Bizard E, et al. (2008) A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth. Development 135: 1427–1437.G. LefevreV. MichelD. WeilL. LepelletierE. Bizard2008A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth.Development13514271437
  19. 19. Kazmierczak P, Sakaguchi H, Tokita J, Wilson-Kubalek EM, Milligan RA, et al. (2007) Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature 449: 87–91.P. KazmierczakH. SakaguchiJ. TokitaEM Wilson-KubalekRA Milligan2007Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.Nature4498791
  20. 20. Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS, et al. (2004) Cadherin 23 is a component of the tip link in hair-cell stereocilia. Nature 428: 950–955.J. SiemensC. LilloRA DumontA. ReynoldsDS Williams2004Cadherin 23 is a component of the tip link in hair-cell stereocilia.Nature428950955
  21. 21. Di Palma F, Holme RH, Bryda EC, Belyantseva IA, Pellegrino R, et al. (2001) Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D. Nat Genet 27: 103–107.F. Di PalmaRH HolmeEC BrydaIA BelyantsevaR. Pellegrino2001Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet27103107
  22. 22. Gibson F, Walsh J, Mburu P, Varela A, Brown KA, et al. (1995) A type VII myosin encoded by the mouse deafness gene shaker-1. Nature 374: 62–64.F. GibsonJ. WalshP. MburuA. VarelaKA Brown1995A type VII myosin encoded by the mouse deafness gene shaker-1.Nature3746264
  23. 23. Johnson KR, Gagnon LH, Webb LS, Peters LL, Hawes NL, et al. (2003) Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Hum Mol Genet 12: 3075–3086.KR JohnsonLH GagnonLS WebbLL PetersNL Hawes2003Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene.Hum Mol Genet1230753086
  24. 24. Wilson SM, Householder DB, Coppola V, Tessarollo L, Fritzsch B, et al. (2001) Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice. Genomics 74: 228–233.SM WilsonDB HouseholderV. CoppolaL. TessarolloB. Fritzsch2001Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.Genomics74228233
  25. 25. Kikkawa Y, Shitara H, Wakana S, Kohara Y, Takada T, et al. (2003) Mutations in a new scaffold protein Sans cause deafness in Jackson shaker mice. Hum Mol Genet 12: 453–461.Y. KikkawaH. ShitaraS. WakanaY. KoharaT. Takada2003Mutations in a new scaffold protein Sans cause deafness in Jackson shaker mice.Hum Mol Genet12453461
  26. 26. Ernest S, Rauch GJ, Haffter P, Geisler R, Petit C, et al. (2000) Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness. Hum Mol Genet 9: 2189–2196.S. ErnestGJ RauchP. HaffterR. GeislerC. Petit2000Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness.Hum Mol Genet921892196
  27. 27. Seiler C, Finger-Baier KC, Rinner O, Makhankov YV, Schwarz H, et al. (2005) Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision. Development 132: 615–623.C. SeilerKC Finger-BaierO. RinnerYV MakhankovH. Schwarz2005Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision.Development132615623
  28. 28. Sollner C, Rauch GJ, Siemens J, Geisler R, Schuster SC, et al. (2004) Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells. Nature 428: 955–959.C. SollnerGJ RauchJ. SiemensR. GeislerSC Schuster2004Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells.Nature428955959
  29. 29. Kiehart DP, Franke JD, Chee MK, Montague RA, Chen TL, et al. (2004) Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA. Genetics 168: 1337–1352.DP KiehartJD FrankeMK CheeRA MontagueTL Chen2004Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.Genetics16813371352
  30. 30. Todi SV, Franke JD, Kiehart DP, Eberl DF (2005) Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila. Curr Biol 15: 862–868.SV TodiJD FrankeDP KiehartDF Eberl2005Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila.Curr Biol15862868
  31. 31. Todi SV, Sivan-Loukianova E, Jacobs JS, Kiehart DP, Eberl DF (2008) Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development. PLoS ONE 3: e2115.SV TodiE. Sivan-LoukianovaJS JacobsDP KiehartDF Eberl2008Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development.PLoS ONE3e2115
  32. 32. D'Alterio C, Tran DD, Yeung MW, Hwang MS, Li MA, et al. (2005) Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli. J Cell Biol 171: 549–558.C. D'AlterioDD TranMW YeungMS HwangMA Li2005Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli.J Cell Biol171549558
  33. 33. Schlichting K, Wilsch-Brauninger M, Demontis F, Dahmann C (2006) Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells. J Cell Sci 119: 1184–1195.K. SchlichtingM. Wilsch-BrauningerF. DemontisC. Dahmann2006Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells.J Cell Sci11911841195
  34. 34. Spradling AC (1993) Developmental Genetics of Oogenesis. In: Bate M, Martinez Arias A, editors. The Development of Drosophila melanogaster. New York: Cold Spring Harbor Laboratory Press. pp. 1–70.AC Spradling1993Developmental Genetics of Oogenesis.M. BateA. Martinez AriasThe Development of Drosophila melanogasterNew YorkCold Spring Harbor Laboratory Press170
  35. 35. Williams DS (2008) Usher syndrome: animal models, retinal function of Usher proteins, and prospects for gene therapy. Vision Res 48: 433–441.DS Williams2008Usher syndrome: animal models, retinal function of Usher proteins, and prospects for gene therapy.Vision Res48433441
  36. 36. Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS (1995) Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc Natl Acad Sci U S A 92: 9815–9819.T. HassonMB HeintzelmanJ. Santos-SacchiDP CoreyMS Mooseker1995Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B.Proc Natl Acad Sci U S A9298159819
  37. 37. Lu H, Bilder D (2005) Endocytic control of epithelial polarity and proliferation in Drosophila. Nat Cell Biol 7: 1232–1239.H. LuD. Bilder2005Endocytic control of epithelial polarity and proliferation in Drosophila.Nat Cell Biol712321239
  38. 38. Fung S, Wang F, Chase M, Godt D, Hartenstein V (2008) Expression profile of the cadherin family in the developing Drosophila brain. J Comp Neurol 506: 469–488.S. FungF. WangM. ChaseD. GodtV. Hartenstein2008Expression profile of the cadherin family in the developing Drosophila brain.J Comp Neurol506469488
  39. 39. Zartman JJ, Kanodia JS, Yakoby N, Schafer X, Watson C, et al. (2009) Expression patterns of cadherin genes in Drosophila oogenesis. Gene Expr Patterns 9: 31–36.JJ ZartmanJS KanodiaN. YakobyX. SchaferC. Watson2009Expression patterns of cadherin genes in Drosophila oogenesis.Gene Expr Patterns93136
  40. 40. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.SF AltschulTL MaddenAA SchafferJ. ZhangZ. Zhang1997Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res2533893402
  41. 41. Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A 95: 5857–5864.J. SchultzF. MilpetzP. BorkCP Ponting1998SMART, a simple modular architecture research tool: identification of signaling domains.Proc Natl Acad Sci U S A9558575864
  42. 42. Schlichting K, Demontis F, Dahmann C (2005) Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila. Dev Biol 279: 142–154.K. SchlichtingF. DemontisC. Dahmann2005Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila.Dev Biol279142154
  43. 43. Johnston AM, Naselli G, Niwa H, Brodnicki T, Harrison LC, et al. (2004) Harp (harmonin-interacting, ankyrin repeat-containing protein), a novel protein that interacts with harmonin in epithelial tissues. Genes Cells 9: 967–982.AM JohnstonG. NaselliH. NiwaT. BrodnickiLC Harrison2004Harp (harmonin-interacting, ankyrin repeat-containing protein), a novel protein that interacts with harmonin in epithelial tissues.Genes Cells9967982
  44. 44. Cavener DR (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 15: 1353–1361.DR Cavener1987Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates.Nucleic Acids Res1513531361
  45. 45. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.AH BrandN. Perrimon1993Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development118401415
  46. 46. Rorth P (1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci U S A 93: 12418–12422.P. Rorth1996A modular misexpression screen in Drosophila detecting tissue-specific phenotypes.Proc Natl Acad Sci U S A931241812422
  47. 47. Wu CT, Budding M, Griffin MS, Croop JM (1991) Isolation and characterization of Drosophila multidrug resistance gene homologs. Mol Cell Biol 11: 3940–3948.CT WuM. BuddingMS GriffinJM Croop1991Isolation and characterization of Drosophila multidrug resistance gene homologs.Mol Cell Biol1139403948
  48. 48. Lasko PF, Pardue ML (1988) Studies of the genetic organization of the vestigial microregion of Drosophila melanogaster. Genetics 120: 495–502.PF LaskoML Pardue1988Studies of the genetic organization of the vestigial microregion of Drosophila melanogaster.Genetics120495502
  49. 49. Pignoni F, Zipursky SL (1997) Induction of Drosophila eye development by decapentaplegic. Development 124: 271–278.F. PignoniSL Zipursky1997Induction of Drosophila eye development by decapentaplegic.Development124271278
  50. 50. Johnson K, Grawe F, Grzeschik N, Knust E (2002) Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration. Curr Biol 12: 1675–1680.K. JohnsonF. GraweN. GrzeschikE. Knust2002Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration.Curr Biol1216751680
  51. 51. Oda H, Uemura T, Harada Y, Iwai Y, Takeichi M (1994) A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev Biol 165: 716–726.H. OdaT. UemuraY. HaradaY. IwaiM. Takeichi1994A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion.Dev Biol165716726