Lefebvre et al. 10.1073/pnas.0610822104. |
Fig. 5. Reduced b-dystroglycan protein levels but axial muscle structure is maintained in dag1-MO injected larvae. In dag1-MO injected larvae at 96 hpf, b-DG immunoreactivity is reduced but muscle integrity is maintained. (A-C) b-DG staining of somitic muscle of wild-type uninjected (A), dag1-MO injected wild-type (B), and dag1-MO injected unplugged mutant larvae (C). Compared to wild-type larvae (D), trunk morphology is normal in dag1 wild-type morphant (E) and unplugged morphant larvae (F), with the exception of mild edema (black arrowhead) and epithelial defects in the tail (black arrow). Histological cross-sections of muscle reveal intact muscle fiber organization and cross-striations in dag1 wild-type morphant (H) and dag1 unplugged morphant (I), similar to uninjected wild-type larvae (G). (J) Western blot analysis of b-DG protein levels in uninjected and dag1-MO injected larvae. Protein was extracted from pools of 5 larvae at 96hpf, collected during two independent experiments. W, wild type; U, unplugged; +, dag1-MO-injected; -, uninjected.
Table 1. Morpholino-mediated knockdown of dag-1 on AChR cluster number, area, and synaptic localization
Group | AChR cluster number | AChR cluster area, mm2 | Percentage of AChR clusters with SV2-positive localization |
Wild-type 96 hpf uninjected | 24.0 ± 5.2 | 2.3 ± 2.4 | 39.8 ± 3.9 |
Wild-type + dag1-MO | 20.6 ± 3.7 | 2.0 ± 2.2 | 38.9 ± 4.5 |
P value | 0.99 | 0.023 | 0.56 |
unpluggedbr307 uninjected | 17.8 ± 4.9 | 0.8 ± 0.6 | 33.8 ± 5.2 |
unpluggedbr307 + dag1-MO | 2.6 ± 2.4 | 0.3 ± 0.2 | 19.4 ± 18.4 |
P value | <0.0001 | <0.0001 | <0.01 |
Quantification by pixel analysis of AChR clusters in 96-hpf larvae, wild-type or unplugged, and uninjected or dag1-MO-injected.
SI Materials and Methods
Expression Constructs.
UnpFL- and UnpSV1-eGFP/5xMyc fusion constructs: the PCR primers (forward); unpFL2(XhoI), 5'-AAAAACTCGAGGTTCTGATACGAGGCTGACCAAT-3'; unpSV1(XhoI), 5'-AAAAACTCGAGCCATGATCAGGCCTGCAGAC-3'; nonstop(reverse), 5'-AAAAAGAATTCCTCGAGGCTAGCAGAAAGACCAGATTTGAGCATCTGGTCATGC-3' were used to modify the stop codon to generate pBS-unpNS (NS: nonstop). To amplify and clone GFP with NheI sites in frame into pBSunpNS, the following primers were used: EGFPF(NheI): AAAAAAGCTAGCGCCCCCCGAACCACGGGGACGTG and EGFPR(NheI): AAAAAAGCTAGCTTACTTGTACAGCTCGTCCATGCC.A 5x myc tag or eGFP sequence was cloned into the NheI site in pBS-unpsvNS or pBS-unpflNS to form pBS- UnpSV/FL-eGFP/5xmyc. Unpfl/sv-eGFP/5xmyc were excised by XhoI and cloned into XhoI- digested pCS2+. To construct the zebrafish myogenin::Unpfl-5xmyc and myog::Unpsv-eGFP, the pBS-zfmyog::eGFP reporter plasmid (1) was digested at the XbaI site, then treated with Klenow-mediated nucleotide filling to blunt 5' end, and then digested at the NotI site. Unpfl- 5xmyc or Unpsv-eGFP were released from pCS2+ vector by PstI and NotI, and cloned into pBS-zfmyog to create pBS-zfmyog::unpfl-5xmyc and pBS-zfmyog::unpsv-egfp. For the zebrafish muscle a-actin::dag1-5xmyc construct, dag1 cDNA was amplified from a zebrafish BAC (BC099987), beginning at the translation start site to prevent targeting by the dag1 antisense morpholino, using primers: EcoRI-dag1-F: GGAATTCGCCACCATGCGCAATAAACTCAGAG and dag1-NheI-XbaI-R: CTCTAGAGCTAGCGGGTGGCACGTAAGGGG. The dag1 cDNA was subcloned into the EcoR1 and XbaI sites of the pCS2+ vector, and then inserted into the EcoRI and NotI sites of the zebrafish muscle a-actin::gfp plasmid (2), to give rise to muscle a-actin::dag1 plasmid. Plasmids were diluted in injection buffer and injected into one-cell embryos, as described previously (Downes and Granato, 2004).
Immunostaining and a-Bungarotoxin Labeling.
Seventeen- to 120-hpf embryos were anesthetized (0.01% Tricaine), fixed in 4% paraformaldehyde with 1% DMSO for three hours at room temperature or overnight at 4°C, and then washed several times in 0.1M phosphate buffer pH 7.4. Immunostainings and labeling of AChRs with AlexaFluor conjugated a-bungarotoxin (Molecular Probes, Eugene, OR) were performed as described (3). AChR clustering was quantified by counting muscle hemisegments (somitic segments 5-15) in which most or all myofibers lacked AChRs clusters. Those hemisegments quantified as 'strongly reduced' typically contained <5 AChR clusters. For AChR cluster quantification in unplugged and dag1 morphant, 4-dpf larvae were bisected and placed in individual tubes for staining of the the rostral trunk with a-BTX/SV2 and caudal trunk with a-BTX/b-DG. The following primary antibodies and dilutions were used: znp-1 (1:200; (4); Antibody Facility, University of Oregon); SV2 (1:50, Developmental Studies Hybridoma Bank, University of Iowa); b-DG (1:50, Novacastra); laminin-1 (1:200; Sigma L-9393). Antibodies were visualized with corresponding AlexaFluor 488, 594 or 633 conjugated secondary antibodies (1:500; Molecular Probes, Eugene, OR). Fluorescent embryos were immersed and mounted in Vectashield mounting medium (Vector Laboratories). Embryos were viewed by using epifluorescence on a Zeiss Axioplan microscope (Zeiss, Thornwood, NY) or Leica MZFLIII stereomicroscope (Bannockburn, IL). Images were obtained by using a laser-scanning LSM510 confocal microscope (Zeiss).Histological Section of Trunk Muscle Tissue.
Four- to 5-dpf larvae were fixed in 4% paraformaldehyde, washed in PBST, and embedded in glycol methacrylate (JB4, Polysciences Inc., Warrington PA). Five-micrometer sections were cut by using a Leica RM2155 microtome and stained in methylene blue/azure II. Sections were analyzed with a Zeiss Axioscope compound microscope.Pixel Quantification and Statistical Analysis.
Eight 96 hpf larvae, from uninjected and dag1-MO-injected wild-type and unplugged pools obtained from 2 different experiments, were analyzed. For each larva, three different hemisegment fields (3037.5 mm area) of optical sections obtained from similar medio-lateral and rostral-caudal levels (somitic hemisegments 7 g) were analyzed. For scanning of images, laser intensity levels and detector gain and offset were adjusted so that no pixel values were saturated in regions analyzed. Confocal images were thresholded, and the number, area of individual clusters and synaptic localization of AChRs and SV2-positive nerve terminals were determined by using ImageJ software (NIH). Cluster number was compared by using the Mann-Whitney or Kruskal-Wallis ANOVA test. Cluster area values were plotted as cumulative histograms for each condition, and distributions were compared by using the Kolmolgorov-Smirnov two-sample test.Western Blotting.
Western blotting protocol was modified from ref. 5. Unfixed 96 hpf larvae were flash frozen and stored at -80°C. Protein from pools of 5 larvae was prepared by homogenization with pestle in suspension buffer (100 mM NaCL, 10 mM Tris·Cl pH 7.6, 1 mM EDTA pH 8.0, 1/50 dilution of protease inhibitor mixture (Sigma), followed by the addition of an equal volume of 2x sample buffer (100 mM Tris·Cl pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol). Samples were placed in an 80°C water bath for 10 min, followed by centrifugation at 10,000 ´ g for 10 min. Protein concentrations were determined (Bradford Protein Assay, BioRad) and volumes corresponding to equal protein quantities (roughly one larvae) were loaded. Proteins were separated by SDS-PAGE (10%) gradient and transferred to a nitrocellulose membrane (Hybond-P, Amersham Pharmacia Biosciences). Membranes were incubated in blocking buffer, and probed with primary antibodies: b-DG (1:200; Novacastra). After washing, blots were incubated with appropriate HRP-conjugated secondary antibody (Jackson Immunoresearch), and then detected by ECL Plus chemiluminescent detection system (Amersham Pharmacia Biosciences). Membranes were stripped in stripping buffer (2% SDS, 100 mM b-mercaptoethanol, 50 mM Tris·HCl pH 6.8) for 30 min at 50°C and probed with anti-b-actin antibody (1:40,000).1. Du SJ, Gao J, Anyangwe V (2003) Comp Biochem Physiol B Biochem Mol Biol 134:123-134.
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5. Sollner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C, Nicolson T (2003) Science 302:282-286.