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First published online 14 December 2005
doi: 10.1242/dev.02195

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The conserved oligomeric Golgi complex acts in organ morphogenesis via glycosylation of an ADAM protease in C. elegans

¹ RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.
² PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.

View larger version (64K): [in a new window]   Fig. 1. Wild-type and mutant gonad morphology. (A) Schematic diagrams of gonad morphology in the wild-type hermaphrodite (left) and the phases of DTC migration (right). Ventral and dorsal body wall muscles are shown in blue. Gonad arms (yellow) are surrounded by basement membranes (green). The uncolored part corresponds to the lateral hypodermis. Two DTCs (red) are generated at the anterior and posterior ends of the gonad primordium, located at the ventral mid-body, and migrate in a U-shaped pattern during gonad development. DTC migration comprises three phases: the initial migration on the ventral body wall muscle (phase 1), the ventral-to-dorsal migration along the lateral hypodermis (phase 2) and the migration along the dorsal body wall muscle (phase 3) (Hedgecock et al., 1987; Su et al., 2000). (B-G) Nomarski images of the posterior gonad arms of wild-type (B), mig-17(k174) (C), cogc-3(k181) (D,E) and cogc-1(k179) hermaphrodites (F,G). The gonads exhibit a meandering morphology in cogc-3(k181) and cogc-1(k179) mutants. The migration paths of DTCs are depicted by orange lines and arrowheads. Anterior is leftwards. Dorsal is towards the top. A, anterior; P, posterior; D, dorsal; V, ventral. Scale bar: 25 µm. (H) Percentages of anterior (left) and posterior (right) gonad arms that underwent normal or defective DTC migration. The migration defects were scored as the earliest phase during which a defect was evident. n=120 for each experiment. (I) Genetic interactions of mig-17 with cogc-3 or cogc-1. Percentages of anterior and posterior gonad arms having phase 2 migration defects. n=120 for each experiment. The error bars represent the mean±s.d. The values were calculated from the data collected from the first and second half of the animals examined.

View larger version (66K): [in a new window]   Fig. 2. Genetic mapping and molecular cloning of cogc-3(k181) and cogc-1(k179) mutants. (A) (Top) Genetic map of cogc-3 and cogc-1 loci. (Middle) The exon and intron structures of cogc-3 and cogc-1 genes. Arrowheads indicate the positions of transposon insertions. (Bottom) The PCR fragments used for injection rescue experiments. (B) Homology between COGC-3 and Homo sapiens (Hs) Sec34 (COG-3). (C) Homology between COGC-1 and Hs ldlBp (COG-1). Black boxes indicate identical amino acids. (D) Domain structures of COGC-3 and COGC-1. Conserved domains are shown in colored boxes with percentages of amino acid identity in human and C. elegans homologs. Amino acid positions in C. elegans proteins are shown in parentheses. The gene products of cogc-3 and cogc-1 correspond to the GenBank entries AB212858 and AB212859, respectively.

View larger version (85K): [in a new window]   Fig. 3. RNA interference of components of the COG complex. Schematic diagrams of the COG complex in mammals (A) and C. elegans (B). Nomarski images of the posterior gonad arms of animals treated with RNAi directed against cogc-1 (C), cogc-2 (D), cogc-3 (E) or cogc-4 (F). Anterior is leftwards. Dorsal is towards the top. Scale bar: 25 µm. The migration paths of DTCs are depicted by orange lines and arrowheads. (G) Percentages of anterior and posterior gonad arms with DTC migration defects in RNAi knockdowns of COG components in the wild-type background. n=30 for each experiment. The following cDNAs were used for RNAi analysis of COG complex components: yk31b12 corresponds to cogc-1; yk324g6 to C06G3.10/cogc-2; yk863b10 to cogc-3; yk402e5 to Y51H7C.6/cogc-4; yk621c10 to C43E11.1/cogc-5; yk29e8 to K07C11.9/cogc-6; yk730h8 to W01B6.9/cogc-7; and yk26e9 to R02D3.2/cogc-8. The error bars represent the mean±s.d. The values were calculated from the data collected from the first and second half of the animals examined.

View larger version (46K): [in a new window]   Fig. 4. COGC-3 and COGC-1 immunoblotting. Western blot analysis of COGC-3 (A) and COGC-1 (B). Protein lysates prepared from wild-type, cogc-3 or cogc-1 worms were immunoblotted with anti-COGC-3 or anti-COGC-1. -tubulin was used as a loading control. (C,D) Co-immunoprecipitation experiments. Protein lysate prepared from wild-type worms was immunoprecipitated with anti-COGC-3 or normal rabbit IgG (negative control) and then immunoblotted with anti-COGC-3 (C) or anti-COGC-1 (D).

View larger version (76K): [in a new window]   Fig. 5. Expression of COGC-3. (A-C) Immunolocalization of COGC-3 in a cross-section of a wild-type L4 larva. The section was stained with anti-COGC-3 (red), fluorescein phalloidin (green) and DAPI (blue). (A) Merged image. (B) Anti-COGC-3 image. (C) Schematic drawing of the positions of tissues. Arrowhead indicates the perinuclear localization of COGC-3 in a muscle cell. The muscle cells contain actin filaments stained by fluorescein phalloidin apically and the nuclei basally. (D) Localization of COGC-3 in a cogc-3(k181) L4 larva. No COGC-3 signal was detected. Dorsal is towards the top. Scale bar: 12.5 µm. (E-J) Whole-mount immunohistochemistry with COGC-3 antibody. Antibody staining patterns (EG,I) and corresponding schematic drawings (F,H,J) of wild-type L4 larvae. (E,F) Pharynx, (G,H) vulva and (I,J) seam cell. Dorsal is towards the top. Anterior is leftwards. Scale bar: 25 µm.

View larger version (67K): [in a new window]   Fig. 6. Immunocytochemistry of COGC-3. (A-C) Confocal images of a body wall muscle cell of an L4 larva transgenic for mig-23::GFP. (A) Anti-COGC-3, (B) anti-GFP and (C) a merged image. Some of the COGC-3 signals colocalized with MIG-23-GFP (arrowheads). (D-F) Confocal images of a body wall muscle cell of an L4 larva transgenic for YFP::TRAM. (D) Anti-COGC-3, (E) anti-GFP and (F) a merged image. YFP-TRAM and COGC-3 signals colocalized at the perinuclear region (small arrows). (G,H) Confocal images of MIG-23-GFP in body wall muscle cells in cogc-3(k181) L4 larvae. (G) Anti-COGC-3 and (H) anti-GFP. The number and intensity of MIG-23-GFP signals were reduced. A similar result was obtained in cogc-1(k179) (data not shown). (I-N) Confocal images of mRFP-SP12 (I,L) and MIG-23-GFP (J,M) and merged images (K,N) in body wall muscle cells in wild-type (I-K) and cogc-3(k181) (L-N) L4 larvae. MIG-23-GFP partially colocalizes with mRFP-SP12 in wild type, but not in cogc-3 mutants. The boundaries of the muscle cells are depicted by broken white lines. Scale bars: 25 µm. (O) Western blot of endogenous MIG-23. Protein lysates prepared from each strain were immunoblotted with anti-MIG-23 and anti--tubulin.

View larger version (48K): [in a new window]   Fig. 7. MIG-17 glycosylation and localization in cogc-3(k181) and cogc-1(k179) mutants. (A) Percentages of anterior and posterior gonad arms with DTC migration defects in cogc-3(k181) worms carrying extrachromosomal transgenic arrays. cogc-3 expression under the muscle-specific promoter unc-54 rescues the DTC phenotype of cogc-3 mutants. Both phase 2 and phase 3 defects were partially rescued (data not shown). The same transgenic array caused no DTC migration defects in wild-type background, indicating the partial rescue is not due to the overexpression of COGC-3. At least two independent lines were scored for transgenics, yielding similar results. Data for representative animals are shown. n=120 for each experiment. The error bars represent the mean±s.d. The values were calculated from the data collected from the first and second half of the animals examined. (B) N-glycosylation of MIG-17. Worm lysates were immunoprecipitated and immunoblotted with anti-GFP. The signals correspond to proforms of MIG-17-GFP. MIG-17-GFP in cogc-3(k181) and cogc-1(k179) mutants migrate further than wild-type MIG-17-GFP. Brackets indicate smearing of the bands in cogc-3(k181) and cogc-1(k179). The underglycosylation of MIG-17-GFP is more prominent (arrowhead) in cogc-1(k179). Part of each immunoprecipitate was treated with PNGase F (N-glycanase) and immunoblotted with anti-GFP. (C) Gonadal localization of MIG-17-GFP in cross sections (upper), and corresponding schematic diagrams (lower). Sections were stained with anti-GFP (red), fluorescein phalloidin (green) and DAPI (blue). The circular arrangement of nuclei in wild-type, cogc-3(k181) and cogc-1(k179) animals correspond to germ line nuclei of the gonads. Arrowheads indicate the gonadal localization of MIG-17-GFP to the surface of distal (upper) and proximal (lower) gonad arms in wild type. The gonadal localization is shown in red circles in the diagram. Dorsal is towards the top. Scale bar: 12.5 µm. (D) Quantitative analysis of MIG-17-GFP localization in wild type and mutants. n=60 for each experiment.

View larger version (71K): [in a new window]   Fig. 8. Secretion of MIG-17-Venus. (A-F) Uptake of ssGFP (A-C) and MIG-17-Venus (D-F) secreted from muscles by wild type, cogc-3 and cogc-1 coelomocytes. Fluorescence images are shown beneath the Nomarski images of the same coelomocytes. All images were captured under the same exposure condition. Scale bar: 10 µm.

View larger version (20K): [in a new window]   Fig. 9. Ectopic expression of MIG-17 and COGC-3 in touch neurons. (Top) Schematic diagram of touch neurons (red) on the right side of the worm. (Bottom) Percentages of anterior and posterior gonad arms with phase 2 DTC migration defects in cogc-3(k181) and strains expressing cogc-3 and/or mig-17::GFP in touch neurons. More than two independent lines were scored for transgenics, yielding similar results. Data for representative animals are shown. n=120 for each experiment. The error bars represent the mean±s.d. The values were calculated from the data collected from the first and second half of the animals examined.