Drosophila Gp150 is required for early ommatidial development through modulation of Notch signaling
2002; Springer Nature; Volume: 21; Issue: 5 Linguagem: Inglês
10.1093/emboj/21.5.1074
ISSN1460-2075
Autores Tópico(s)Animal Genetics and Reproduction
ResumoArticle1 March 2002free access Drosophila Gp150 is required for early ommatidial development through modulation of Notch signaling Michael Fetchko Michael Fetchko Department of Biochemistry and Molecular Biology, University Park, PA, 16802 USA Search for more papers by this author Wei Huang Wei Huang Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Ying Li Ying Li Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Zhi-Chun Lai Corresponding Author Zhi-Chun Lai Department of Biochemistry and Molecular Biology, University Park, PA, 16802 USA Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Michael Fetchko Michael Fetchko Department of Biochemistry and Molecular Biology, University Park, PA, 16802 USA Search for more papers by this author Wei Huang Wei Huang Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Ying Li Ying Li Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Zhi-Chun Lai Corresponding Author Zhi-Chun Lai Department of Biochemistry and Molecular Biology, University Park, PA, 16802 USA Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Author Information Michael Fetchko1, Wei Huang2, Ying Li2 and Zhi-Chun Lai 1,2 1Department of Biochemistry and Molecular Biology, University Park, PA, 16802 USA 2Department of Biology, The Pennsylvania State University, University Park, PA, 16802 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1074-1083https://doi.org/10.1093/emboj/21.5.1074 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cellular signaling activities must be tightly regulated for proper cell fate control and tissue morphogenesis. Here we report that the Drosophila leucine-rich repeat transmembrane glycoprotein Gp150 is required for viability, fertility and development of the eye, wing and sensory organs. In the eye, Gp150 plays a critical role in regulating early ommatidial formation. Gp150 is highly expressed in cells of the morphogenetic furrow (MF) region, where it accumulates exclusively in intracellular vesicles in an endocytosis-independent manner. Loss of gp150 function causes defects in the refinement of photoreceptor R8 cells and recruitment of other cells, which leads to the formation of aberrant ommatidia. Genetic analyses suggest that Gp150 functions to modulate Notch signaling. Consistent with this notion, Gp150 is co-localized with Delta in intracellular vesicles in cells within the MF region and loss of gp150 function causes accumulation of intracellular Delta protein. Therefore, Gp150 might function in intracellular vesicles to modulate Delta–Notch signaling for cell fate control and tissue morphogenesis. Introduction Development of multicellular organisms requires not only precise cell fate specification, but also proper patterning of differentiating cells. The Drosophila compound eye provides a model system to address how cells are specified and organized. During the third instar larval stage, a dorso-ventral indentation called the morphogenetic furrow (MF) moves from the posterior of the eye tissue to the anterior, and formation of ommatidial clusters is initiated in areas immediately posterior to the MF (for review see Wolff and Ready, 1993). Expression of the proneural basic helix–loop–helix (bHLH) atonal (ato) gene is first induced by Hedgehog signaling in a stripe of cells just anterior to the MF (for review see Heberlein and Moses, 1995; Treisman and Heberlein, 1998). Within the MF, ato expression is restricted to regularly spaced proneural clusters of cells. Only one cell from each proneural cluster continues to express ato and becomes the R8 photoreceptor (R), which then initiates further recruitment of other R cells for ommatidial construction (Jarman et al., 1994, 1995; Baker et al., 1996; Dokucu et al., 1996; Baonza et al., 2001). Notch (N) signaling initially plays a positive role in establishing high levels of ato in cells anterior to the MF (Baker and Yu, 1997; Li and Baker, 2001) by down-regulating Hairy (H) and Extramacrochaetae (Emc), two repressors of ato (Baonza and Freeman, 2001). Subsequently, N-mediated lateral inhibition is required for the refinement of ato expression to regularly spaced individual R8 precursor cells (Parks et al., 1995; Baker et al., 1996; Dokucu et al., 1996). The Drosophila homolog of the vertebrate epidermal growth factor receptor (DER) functions to regulate R8 spacing as well. DER signaling acts non-autonomously to inhibit ato expression in cells anterior and lateral to the proneural clusters to generate regularly spaced proneural clusters (Chen and Chien, 1999; Baonza et al., 2001). Also, a glycoprotein Scabrous (Sca) might be responsible for anterior and lateral repression of Ato, as Sca is produced in proneural clusters and can be secreted (Baker and Zitron, 1995; Lee et al., 1996). Sca associates with N in vivo and can stabilize N proteins at the cell surface (Powell et al., 2001). In precursor cells located more posterior to the MF, N signaling is required for restricting cellular competence to respond to receptor tyrosine kinase (RTK)-mediated inductive signaling for R cell specification. Supporting this idea, several E(spl) proteins are prominently expressed in the basally located nuclei of the precursor cells (Baker et al., 1996). A deletion of a subset or all of the bHLH genes in the E(spl) complex results in a severe neurogenic phenotype in the posterior region of eye discs (Treisman et al., 1997; Ligoxygakis et al., 1998). Thus, coordinated action of inductive RTK signaling (for review see Freeman, 1997) and N-mediated lateral inhibition (for review see Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000) is critical for the sequential recruitment of R cells into the developing ommatidia. Here we report the identification and isolation of loss-of-function mutations in the Drosophila gp150 gene, which encodes a leucine-rich repeat (LRR) protein that is required for viability, fertility and proper development of the eye, wing and sensory organs. In the eye, removal of gp150 function causes defects in the refinement of R8 cells and recruitment of other cells, which leads to the formation of fused ommatidia, as well as ommatidia containing too many or too few R cells. We show that Gp150 is expressed at high levels in the MF region, which is consistent with a role of gp150 in early ommatidial development. Moreover, genetic analyses suggested that Gp150 functions to modulate Delta (Dl)–N signaling, and immunostaining experiments showed that Gp150 is co-localized with Dl in intracellular vesicles in cells within the MF region. Gp150 might be involved in facilitating lysosomal delivery of the Dl protein and/or Dl transport to the plasma membrane, as loss of gp150 function causes accumulation of intracellular Dl protein. Gp150 appears to be a resident protein of intracellular vesicles and its localization is not affected in endocytosis-defective cells. Based on these observations, we propose that Gp150 might function in subcellular vesicles to control appropriate intracellular levels of Dl to modulate N signaling. Results Identification and isolation of loss-of-function mutations in the Drosophila gp150 gene, which encodes a LRR transmembrane protein Flies homozygous for two presumably P transposon-induced semi-lethal mutations, l(2)k11107 and l(2)k11120 (Torok et al., 1993), exhibit reduced fertility and abnormal bristle, wing and eye phenotypes. These two allelic recessive mutations were meiotically mapped to the tip of the right arm of the second chromosome. Deficiency mapping further defines the locus of these mutations to the 58D1-5 region. However, no wild-type revertants were recovered by P-element excision (data not shown). Thus, these alleles could have been caused by an imprecise excision of a P-element initially inserted at the locus. To facilitate molecular genetic analysis of this gene, we generated new alleles through ethyl methane sulfonate (EMS) mutagenesis and P-element local hopping (see Materials and methods). Genomic sequences flanking the insertion sites in two of the newly isolated P alleles, P2 and P8, were cloned and used to isolate cDNA clones from a larval eye disc cDNA library. Sequence analysis of full-length cDNAs demonstrated that these cDNA molecules encode a polypeptide identical to the LRR transmembrane glycoprotein Gp150 (Tian and Zinn, 1994). Our sequence analysis, together with data derived from the Drosophila genome project, indicates that the gp150 gene consists of six exons and five introns (Figure 1A). The gp150 open reading frame (ORF) is restricted to the last four exons, which encode a polypeptide of 1051 amino acids. The following evidence demonstrates that mutant alleles of gp150 have been identified. First, in the P8 allele, a P-element is inserted within the second intron of the gp150 transcription unit, 53 bp downstream of the second exon (Figure 1A). The P8 mutant eye discs exhibited a reduction of gp150 expression, as shown by western blotting (Figure 1B) and immunostaining (data not shown). These experiments revealed that P8 is a partial loss-of-function mutation. Furthermore, mutant phenotypes in four out of six newly isolated P alleles can be frequently reverted to wild type by P-element excision (see Materials and methods), indicating that these mutations were indeed caused by P-element insertions. Secondly, a single base change of G to A was found in codon 94 for tryptophan in an EMS allele (gp1504), which results in a nonsense mutation. Thirdly, western blot and immunostaining analyses revealed that the level of Gp150 is either reduced or eliminated by the mutations (Figures 1B and 5B). Lastly, the mutant eye phenotypes can be effectively rescued by overexpression of a wild-type gp150 gene in the developing eye (Figure 3E and F). These loss-of-function mutations were used in this study to reveal a role of gp150 in eye development. Figure 1.Identification of loss-of-function alleles of the gp150 gene. (A) The gp150 gene consists of six exons and five introns with the complete coding sequence restricted within a 4 kb genomic region. The filled boxes represent a complete 3156 bp ORF. Sequences of two genomic regions indicated by the thick horizontal lines were determined to illustrate intron–exon structures of the gp150 transcriptional unit. A PCR method was used to obtain the downstream genomic sequence of gp150 that was not included in the genomic clone. In P8, a P transposon was inserted in the second intron of gp150. Restriction enzymes used for mapping include BamHI (B), EcoRI (E), KpnI (K), SalI (S), XbaI (X) and XhoI (Xh). (B) A western blot was probed with a Gp150 antibody that was made against the N-terminal region (amino acids 5–192) of Gp150. Since no Gp150 protein was detectable in gp1502, gp1503 and gp1504 mutants, these alleles can be considered as molecular nulls. The gp1502 mutation results in the production of a non-functional 90 kDa protein. This is based on observations that gp1502 is recessive and the mutant phenotypes caused by gp1502 are similar to that of gp1503 and gp1504. The 70 kDa Fas I protein served as an internal control and was detected by anti-Fas I (MAb6D8) antibody in all lanes. Download figure Download PowerPoint Figure 2.Loss of gp150 function results in defective wing and sensory organs. Wild-type (A, C and E) and gp1502 mutant (B, D and F) wings are shown. (C) and (E) are enlarged from boxed areas in (A). (D) and (F) are enlarged from the boxed areas in (B). Some bristles are missing (dots) or duplicated (asterisks) in the wing margin (D). Arrows point to some ectopic bristles in the second and third veins (D and F) and the arrowhead identifies an enlarged ‘Delta-like’ vein structure (F). SEM images of wild-type (G and I) and gp1501 mutant flies (H and J) are shown to illustrate phenotypes in ocellar bristles (G and H) and sensory organs over the notum (I and J). Multiple socket cells are often developed in sensory organs (circled in H and indicated by an arrowhead in J). The external shaft is often missing or duplicated (indicated by an arrow in J). Moreover, there are cases where both socket and shaft cells are absent in some sensory organs (H). All other gp150 alleles exhibit similar phenotypes in the wing and sensory organs. Download figure Download PowerPoint Figure 3.Loss of gp150 function causes defective eye development. SEM images (A, C and E) and tangential sections (B, D and F) of adult eyes are presented. (A and B) Wild type. The inset highlights seven R cells arranged in a trapezoidal configuration in each ommatidium and the rhabdomere of R8 cannot be seen in this apical section. (C and D) gp1502 mutants. About 35% (n = 522) of the ommatidia contain either too many or too few R cells. The arrow indicates a fused ommatidium. At basal levels, ommatidia with multiple R8-like cells were observed (data not shown). Other gp150 alleles exhibit similar phenotypes. (E and F) In gp1502 GMR-gp150 flies, overexpression of gp150 in the eye effectively rescues the mutant eye phenotype, with >93% (n = 368) of the ommatidia containing a normal complement of R cells. The arrow indicates a mutant ommatidium occasionally seen in this genotype. Anterior is to the left, except (D), in which anterior might be to the top. Download figure Download PowerPoint gp150 is required for viability, fertility and normal development of the wing and sensory organs Loss of gp150 function results in a strong reduction in viability. For instance, >60% of gp1502/gp1503 flies fail to develop to the adult stage, with most of them dying during pupal development (Table I). gp150 mutant females are sterile as they fail to lay eggs and the fertility of gp150 mutant males is also strongly reduced (data not shown). In gp150− wings, the size and overall morphology appear normal (Figure 2B). However, closer examination revealed that some dorsal margin bristles are missing or duplicated and ectopic bristles can be found in the distal region of the second vein (Figure 2D). Moreover, the distal end of the third vein is enlarged to form a ‘Delta-like’ structure (Figure 2F). Figure 4.Loss of gp150 function causes defects in early ommatidial development. Wild-type (A, C, E, G, I and K) and gp1502/gp1503 mutant (B, D, F, H, J and L) third instar eye discs are shown. (A and B) Phalloidin staining. (C and D) Anti-Ato antibody staining. Arrows in (B and D) point to closely located ommatidia. (E and F) Anti-Boss antibody staining. Arrows indicate ommatidia containing more than one Boss-positive R8 cells. (G and H) Double staining for Hairy and Elav. The arrow points to an oversized ommatidium near the furrow. (I and J) Anti-BarH1 antibody staining. (K and L) Anti-Cut antibody staining. The circle indicates an ommatidium missing a cone cell. Arrowheads identify the location of the MF in (A–H). Anterior is to the left in all panels. Download figure Download PowerPoint Table 1. Gp150 is required for viability 1 2 3 4 Df(2R)02311 1 61 (n = 633) 75 (n = 747) 73 (n = 221) 61 (n = 334) 2 61 (n = 526) 43 (n = 319) 46 (n = 319) 3 93 (n = 476) 63 (n = 331) 4 62 (n = 355) 1, 2, 3 and 4 correspond to gp1501, gp1502, gp1503 and gp1504, respectively. For example, from a cross between gp1502/CyO and gp1503/CyO, 61% of the predicted gp1502/gp1503 mutants fail to develop to the adult stage. n = total number of adult flies scored. During normal bristle development, a single sensory organ precursor (SOP) cell divides to produce two daughter cells, IIa and IIb, which divide and generate a trichogen (shaft) and a tormogen (socket), a neuron, a thecogen (sheath) and a glial cell, respectively (for review see Jan and Jan, 1995; Gho et al., 1999). In gp150 mutants, sensory organ development is defective in ocellar, humeral, scutellar and sensory bristles over the notum and dorsal abdomen (Figure 2H and J). In all of the gp1501 mutant flies, one or more ocellars were found to contain multiple socket cells with the external shaft missing (Figure 2H). Such mutant phenotypes implicate cell fate transformation from IIb to IIa and shaft to socket. Interestingly, external cells (socket and shaft) of some sensory organs appeared to be missing (Figure 2H), suggesting a possible cell fate transformation from IIa to IIb. Such mutant phenotypes were observed in other sensory organs as well. For instance, 68% (n = 31) of the anterior scutellars in gp1502/gp1503 flies exhibited no shaft and no socket, 23% with double socket, 3% with one shaft/two sockets and 3% with two shafts/one socket phenotypes. In conclusion, the gp150 function is required for proper development of several distinct tissue types. Loss of gp150 function results in defective ommatidial development We have focused on analyzing gp150 function using the eye system. The wild-type compound eye of Drosophila melanogaster represents an organization of 800 precisely arrayed unit eyes called ommatidia (Figure 3A). Each ommatidium contains 12 accessory cells and eight R cells arranged in a trapezoidal configuration (Figure 3B). Compared with the wild type, gp150 mutant eyes are rough. Many ommatidia are irregular in shape and size and contain either too many or too few R cells. Also, fused ommatidia are often visible (Figure 3C and D). These results indicate that gp150 is required for normal ommatidial development. Clonal analysis revealed essentially the same mutant phenotypes as those observed in gp150 homozygous mutants (data not shown). To reveal the developmental basis of the eye defects, gp150 mutant third instar eye discs were examined. In wild-type eye discs, phalloidin staining outlines apical cell surfaces and highlights the constricted cells within the MF as well as the cells within the evenly spaced developing ommatidial clusters (Figure 4A). In gp150 mutants, ommatidial clusters are often irregularly spaced and the MF appears broader as the cells within the region are no longer constricted in a narrow stripe (Figure 4B). To elucidate early developmental defects that might lead to the aberrant ommatidial organization, ato expression was examined. ato is essential for R8 specification (Jarman et al., 1994) and is initially expressed in cells anterior to the MF. Within the MF, ato expression is first restricted to proneural groups of cells and then to individual R8 precursor cells (Figure 4C; Jarman et al., 1995). In gp150 mutant eye discs, Ato-positive proneural clusters are abnormally patterned and multiple R8 precursor cells are often closely located (Figure 4D). About 10% (n = 72) of the ommatidia contain multiple Ato-positive R8 precursors that become R8 cells, as inferred from their expression of R8-specific Bride-of-sevenless (Boss) protein (Figure 4F; Kramer et al., 1991). To examine how ommatidia assembly can be affected by gp150 mutations, a neural-specific nuclear marker, Elav (Robinow and White, 1991), was used to highlight all R cells. In the wild type, developing ommatidia are evenly patterned with precursor cells sequentially recruited into ommatidia clusters (Figure 4G). In contrast, ommatidia are irregularly organized in gp150 mutant eye discs. Some oversized ommatidia are seen at early stages of ommatidia assembly (Figure 4H). Thus, recruitment of R cells into the clusters appears to occur prematurely in some clusters. Figure 5.gp150 is expressed at high levels in the MF region in the developing eye. (A) High levels of Gp150 protein were detected in the MF region of wild-type third instar eye discs. Posterior to the furrow, relatively lower levels of Gp150 can be detected around each individual ommatidium. (B) Gp150 expression was not detectable in gp1504 eye discs. (C) High levels of Gp150 expression were detected in all cells behind the furrow in GMR-gp150 eye discs. (D1–3) Double-staining reveals Gp150 (red, D1) and Ato (green, D2) expression in eye discs. A superimposed image from (D1) and (D2) is shown in (D3). (E1–3) Gp150 and Dl proteins were shown to co-localize in subcellular vesicles in the furrow region. (F1–3) In S2 cells co-transfected with pmt-gp150-myc and pmt-Dl, Gp150 and Dl were also shown to co-localize in subcellular vesicles. (G1–3) Heat-treated shits1 mutant third instar eye discs were double stained with anti-Gp150 (red in G1) and anti-Dl (green in G2) antibodies. A superimposed image is shown in (G3). Arrowheads indicate the location of the MF, and anterior is to the left in all panels. Download figure Download PowerPoint To address whether accumulation of oversized ommatidia close to the MF is due to defects in furrow progression, expression of decapentaplegic (dpp) and a bHLH gene hairy (h) was examined, since both of them are involved in regulating furrow progression (Brown et al., 1995; for review see Treisman and Heberlein, 1998). No discernible effect on either h expression anterior to the furrow or dpp expression within the MF was detected in gp150 mutants (Figure 4G and H and data not shown). Moreover, unlike mutations that disrupt furrow initiation and progression (Heberlein et al., 1993; Ma et al., 1993), gp150 mutations do not cause an apparent size reduction of the eye (Figure 3C). These results suggest that gp150 mutations do not disrupt initiation or progression of the MF. To find out whether cells recruited at later stages of ommatidial assembly are affected by gp150 mutations, expression of a homeodomain protein BarH1 was examined. In wild-type eye discs, BarH1 is specifically expressed in R1 and R6 cells (Figure 4I; Higashijima et al., 1992). In the mutant, most R1/R6 cells are irregularly positioned (Figure 4J). Their abnormal arrangement further indicates defective ommatidial organization. Moreover, some ommatidia contain additional or fewer R1/R6 cells (Figure 4J) and cone cells (Figure 4L). Thus, phenotypic analysis of gp150 mutant eye discs indicates that gp150 plays a critical role in regulating ommatidial patterning and assembly in the developing eye. Gp150 is expressed at high levels within cells of the MF region and is localized to intracellular vesicles To characterize the normal pattern of Gp150 expression in the developing eye, Gp150 antibodies were generated and used for immunostaining experiments. Specificity of the Gp150 antibody was confirmed by the absence of staining in gp1504 eye discs (Figure 5B) and the increased staining in GMR-gp150 eye discs (Figure 5C). In wild-type eye discs, cells immediately anterior to and within the furrow region express high levels of Gp150 protein (Figure 5A and D1). Gp150 proteins accumulate in many small vesicles throughout the apical–basal axis in the furrow region. The area with high levels of Gp150 mostly overlaps with cells expressing the proneural gene ato (Figure 5D1–3). This elevated expression of Gp150 in the furrow region is consistent with the notion that Gp150 is required to regulate early ommatidial development. Posterior to the furrow, lower levels of Gp150 were detected around each individual ommatidium (Figure 5A). The Gp150 protein was not detectable in the nucleus or at the plasma membrane of the eye disc cells. To examine how Gp150 might distribute in subcellular compartments, we used a Myc-tagged non-functional Fringe protein as a Golgi-specific marker (Munro and Freeman, 2000). Partial overlap between Gp150- and Fringe-positive vesicles was observed in both sca-Gal4/UAS-fringeADD-myc and GMR-Gal4/UAS-fringeADD-myc eye discs (data not shown). Using a green flourescent protein (GFP)-tagged Drosophila Rab7 protein as a marker for late endosomes (Entchev et al., 2000), we also observed that Gp150 can be partially localized to endosomes (data not shown). Thus, these results support the idea that Gp150 is involved in regulating vesicle trafficking. gp150 genetically interacts with Dl and N The gp150 mutant eye phenotypes closely mimic those caused by defective Dl–N signaling (Cagan and Ready, 1989; Baker et al., 1990; Parks et al., 1995). Thus, Gp150 might be functionally connected to the N pathway. To investigate this possibility, two assays were devised to test genetic interactions between gp150 and Dl and N. In a genetic sensitization assay, the gp150 function is partially removed in gp150P8/gp1502 flies, which allows us to monitor dominant effects of mutations in other genes on gp150 mutant eye phenotypes. Reduction of Dl function strongly enhanced the gp150 mutant eye phenotypes (Figure 6A and B). There was an ∼2- to 3-fold increase in the number of mutant ommatidia in gp150P8/gp1502; Dl−/+ flies compared with the control (Table II). Although not as effective as Dl, N mutations exhibited a similar effect (Table II). These results suggest that Gp150 functions together with Dl and N to regulate ommatidial development. Using the same assay, mutations such as rlS−135, Ras1e1B and Ras1e2F did not effectively alter the gp150 mutant eye phenotypes (data not shown). Both Ras1 and a MAP kinase, which is encoded by the rolled (rl) gene, are key components of the RTK pathway. Figure 6.Genetic interactions between gp150 and Delta. Tangential sections of adult eyes are presented. (A) The gp150P8/gp1502 eye exhibits ∼19% (n = 578) mutant ommatidia. (B) Reduction of Dl function strongly enhances the gp150 mutant phenotypes. Up to 68% (n = 544) abnormal ommatidia, which contain more R cells (arrow), fewer R cells (arrowhead) or apparently fused ommatidial units, were found in gp150P8/gp1502; Dl9P/+ flies. Often rhabdomeres exhibit abnormal morphology (circled asterisk). (C) gp1502/+. The arrow points to an occasionally observed aberrant ommatidium. (D) Dl9P/+ flies, no mutant ommatidia can be found. (E) gp1502/+; Dl9P/+. The circle indicates a fused ommatidium and the arrowhead points to an ommatidium missing an outer R cell. Download figure Download PowerPoint Table 2. gp150 genetically interacts with Notch and Delta Genotype No. of ommatidia examined % mutant gp150P8/gp1502 578 19.2 N55e11/+; gp150P8/gp1502 629 42.3 gp150P8/gp1502; Dl9P/+ 544 67.6 gp150P8/gp1502; Dl6B/+ 460 44.6 Df(2R)02311/+ 645 0.15 gp1502/+ 1051 0.19 N55e11/+ 599 0 N55e11/+; gp1502/+ 769 0.65 Dl9P/+ 655 0 gp1502/+; Dl9P/+ 852 2.70 Dl6B/+ 534 0 gp1502/+; Dl6B/+ 558 0.18 Dl6B/+ (29°C) 539 0.37 gp1502/+; Dl6B/+ (29°C) 330 1.52 For each genotype, 5–9 adult eyes were sectioned and scored. Flies were cultured at 25°C, unless indicated otherwise. Ommatidia that were fused or contained too many or too few rhabdomeres were counted as mutant. Loss-of-function alleles of gp150 exhibit a very weak dominant eye phenotype as a small number of aberrant ommatidia were found in gp150 heterozygotes (Figure 6C; Table II). This is most likely due to haplo-insufficiency since a deficiency chromosome, in which gp150 is completely deleted, exhibits the same phenotype (Table II). In this second assay, Dl mutations also dominantly enhanced gp150 mutant eye phenotypes, as there was a >10-fold increase in the number of mutant ommatidia in gp1502/+; Dl9P/+ flies (Figure 6C–E; Table II). Compared with Dl, N mutations exhibited a weaker enhancing effect (Table II). Altogether, these genetic data suggest that Gp150 is involved in modulating Dl–N signaling in the eye. Dl is co-localized with Gp150 in intracellular vesicles within cells of the MF region To begin to investigate how Gp150 might act to modulate N signaling, we examined whether Dl and Gp150 proteins were co-localized. Cell surface and intracellular expression of Dl within cells of the developing eye has been characterized previously (Parks et al., 1995; Baker and Yu, 1998). Dl protein accumulates to higher levels in endosomal vesicles than on the plasma membrane. Some of these vesicles correspond to multi-vesicular bodies (MVBs) (Parks et al., 1995). Double-labeling experiments showed that Dl was co-localized with Gp150 in intracellular vesicles of cells within the furrow region (Figure 5E1–3). Moreover, Gp150 and Dl proteins were found to be co-localized in 60 out of 66 subcellular vesicles examined in S2 cells that were positive for both Gp150 and Dl expression (Figure 5F1–3). These results suggest that Gp150 may act in late endosomes to modulate the level or activity of Dl protein in the morphogenetic furrow region. Moreover, it is also possible that Gp150 is co-localized with Dl in secretory vesicles or recycling endosomes to facilitate Dl presentation to the plasma membrane. Gp150 accumulation in intracellular vesicles is independent of Dynamin GTPase-mediated endocytosis The accumulation of Dl in intracellular vesicles is dependent on Dynamin-mediated endocytosis, which is critical for N signaling (Parks et al., 1995, 2000). The Drosophila Dynamin GTPase is encoded by the shibire (shi) gene, which is required for endocytosis (Chen et al., 1991; Van der Bliek and Meyerowitz, 1991). In shits1 mutants, Dl proteins fail to be endocytosed and, therefore, accumulate at the plasma membrane (compare Figure 5E2 with G2; Parks et al., 1995). To test whether the accumulation of Gp150 in vesicles is also endocytosis dep
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