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Fig. 1. Dystroglycan depletion affects notochord morphogenesis. (A) Immunodetection of Dg on transverse cryosections from stage 13 to 37 (indicated in the top right of the images). The arrowhead in c indicates a vacuole. (B) Immunoblot of protein extracts from wild-type (WT) embryos or embryos injected with a 5-mispair control morpholino (C-MO) or Dg-MO. (C) Cryosections of embryos co-injected with ras-GFP mRNAs and C-MO (a) or with Dg-MO (b) and then immunostained with antibodies against Dg. In Dg morphants, the Dg staining is lost. (D) The top panels show the embryo phenotypes observed with C-MO or an increasing amount of Dg-MO at stage 32. (a-d) Cryosections treated with Tor70 antibodies showing a dose-dependent effect on the notochord. The arrowhead in c indicates the discontinuity in the notochord sheath. A cell containing Dg-MO (labeled with GFP, arrow and inset in c) escaped from the notochord. (d) At a high amount of Dg-MO, the sheath disappears and the characteristic structure of the notochord is lost. Dotted lines outline the neural tube (NT). (E) Notochord morphogenesis is rescued in Dg morphants through injections of mRNA encoding Dg (Dg-FL mRNA). Dotted lines outline the NT. NC, notochord; S, somite. Scale bars: 25 μm in A,C,Da-d,E; 500 μm in upper panel in D; 10 μm in Dc inset. |
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Fig. 6. Myosin IIA is a Dg partner in notochord. (A) Immunoprecipitation (IPP) using an antibody against β-Dg. The precipitate and supernatant of wild-type (WT) and Dg-MO-injected notochords were subjected to western blotting with antibodies against myosin IIA and β-Dg. (B) Identification of Dg and myosin IIA localization using immunofluorescence confocal microscopy. Colocalization of Dg and myosin IIA is observed at cell cortices at stage 28 in WT embryos. (C) Immunodetection of myosin IIA (a,b) and Tor70 proteins (c,d) in C-MO and Dg-MO notochords. Labeling of myosin IIA was lost in Dg morphants at stage 32. Nuclei are stained with Hoechst (blue). (D) Protein extracts of WT, Dg-MO and myosin IIA-MO embryos were subjected to western blotting with antibodies against myosin IIA, actin and α-tubulin (6% SDS-PAGE) and with an antibody against β-Dg (12% SDS-PAGE). In Dg-MO extracts, Dg expression was strongly decreased, whereas the amount of myosin IIA was comparable to that of controls. Note that the decrease in Dg and myosin is not accompanied by a decrease in α-tubulin (α-Tub) or actin. R, ratios of myosin IIA, β-Dg and actin to α-tubulin normalized to WT. Scale bars: 25 µm in B,C. |
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Fig. 7. Myosin IIA is required for cytoskeletal integrity. (A) Protein extracts of wild-type (WT) or myosin IIA-MO embryos were subjected to western blotting with antibodies against myosin IIA and α-tubulin, which showed the efficiency of the morpholinos. R, ratios of myosin IIA to α-tubulin normalized to WT. (B) Immunodetection of myosin IIA in C-MO and myosin IIA-MO notochords. Labeling of myosin IIA was lost in morphants at stage 28. Dotted lines outline the notochord (NC). (C) Phenotypes of control and myosin IIA morphants at stage 32. (a-d) Immunodetection of Dg and laminin in C-MO embryos and morphants. The notochord diameter and the vacuole size increased. Myosin IIA depletion does not affect Dg and laminin expression, but they do not localize normally. (e,f) Tor70 labeling confirms the increase in the size of the notochord and vacuoles. (D) F-actin and α-tubulin localization, by using confocal microscopy, in control and myosin IIA- and Dg-depleted notochord at stage 32. (a-f) F-actin is revealed by staining with phalloidin. (d-f) Magnification at the cell cortex showing the loss of labeling in morphants; the area corresponds to that highlighted by the white box in the panel above. (g-l) Tubulin is revealed by using an antibody against α-tubulin. (j-l) Magnification at the cell cortex showing the network disruption of α-tubulin; the area corresponds to that highlighted by the white box in the panel above. Dotted lines outline the notochord. S, somite. Scale bars: 25 μm in B,Ca-f,Da-c,Dg-i; 500 μm in C upper panels; 10 μm in Dd-f,Dj-l. |
