Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line
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{"title"=>"Spatio-temporal differences in dystrophin dynamics at mRNA and protein levels revealed by a novel FlipTrap line", "type"=>"journal", "authors"=>[{"first_name"=>"Frederique", "last_name"=>"Ruf-Zamojski", "scopus_author_id"=>"38663442500"}, {"first_name"=>"Vikas", "last_name"=>"Trivedi", "scopus_author_id"=>"56592211300"}, {"first_name"=>"Scott E.", "last_name"=>"Fraser", "scopus_author_id"=>"7201735128"}, {"first_name"=>"Le A.", "last_name"=>"Trinh", "scopus_author_id"=>"35495555900"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"605586027", "doi"=>"10.1371/journal.pone.0128944", "sgr"=>"84939169832", "isbn"=>"1932-6203", "pmid"=>"26083378", "scopus"=>"2-s2.0-84939169832"}, "id"=>"d6a2da62-df1c-34bc-a650-0c479f322f8e", "abstract"=>"Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ct90a). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.", "link"=>"http://www.mendeley.com/research/spatiotemporal-differences-dystrophin-dynamics-mrna-protein-levels-revealed-novel-fliptrap-line", "reader_count"=>13, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Other"=>1, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Other"=>1, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>9, "Medicine and Dentistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>9}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2120450"], "description"=>"<p>(a,b). Dmd-citrine expression in control (a) and <i>dmd</i> morphants (b). Embryos in (a) and (b) were imaged with the same laser power and gain settings. (c) Western-blot of protein extracts from WT (lanes 1, 2) and <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> (lanes 3, 4) embryos with antibody to DMD and tubulin (loading control). Embryos for protein extracts were injected with control morpholino (lanes 1 and 3) and <i>dmd</i> morpholino (lanes 2 and 4, n = 3–6). Arrows point to size difference between Dmd and Dmd-citrine. (d,e) Widefield fluorescent image of 32hpf <i>ct90aGT</i> embryos untreated (d) and treated with heat-shocked at 6-somites stage. Arrow point to defects in Dmd-citrine expression visible between somites 16–25. (f-i) Heat-shocked embryos stained for Dmd (f,g; green) and anti-MHC (h,i; red) at 32hpf. Scale bars (d,e) 50μm (f-i) 20 μm.</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452359, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g006", "stats"=>{"downloads"=>1, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotyping_of_muscular_dystrophy_with_Gt_dmd_citrine_ct90a_/1452359", "title"=>"Phenotyping of muscular dystrophy with <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120377"], "description"=>"<p>(a). Schematic of the FlipTrap vector inserted within <i>dmd</i> locus. Citrine insertion (green) occurs within intron 4–5 in <i>dmd</i> in the <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> trap line (top) and upon translation produces a fluorescently tagged full-length functional Dmd protein (bottom). The exons (blue rectangles), introns (blank line) and domains of the protein are not drawn to scale. (b-e). Wide-field fluorescent images of Dmd-Citrine expression in the <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> line, in the trunk skeletal muscles (b-c) at 24hpf (b) and 32hpf (c); (d-e). Expression in the cranial skeletal muscle at 6dpf (lateral (d) and ventral (e) views). Ventral view showing expression in the skeletal muscle of the branchial arches. Scale bars: (b)-(c) 100μm, (d)-(e) 25μm.</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452304, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g001", "stats"=>{"downloads"=>1, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_FlipTrap_Gt_dmd_citrine_ct90a_line_allows_visualization_of_Dmd_protein_/1452304", "title"=>"FlipTrap <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> line allows visualization of Dmd protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120434"], "description"=>"<p>(a-h). 3D projections of confocal Z-stacks images in developing trunk of <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> embryos from the 20 to 26 somites stages (19–22 hpf), showing expression of Dmd-citrine (a-d, green) and the vital stain BodipyTR methyl ester (e-h, red) from dorsal and lateral view. (i-l) Quantitation of Dmd-citrine fluorescent signal in confocal time-lapse. (i). Kymograph of Dmd-citrine expression color-coded according to developmental stage. The spatial shift in expression appears to be due to tissue growth and expression enhancement over time. (j). Histogram of fluorescent pixel intensities color-coded according to developmental stage with intensity in X-axis and frequency in Y-axis. Plot shows Dmd-citrine expression increasing over time resulting in shift slope along the X-axis (blue to red). The inset shows the same plotted on a log scale to enhance observation. (k). Mean pixel intensity plotted against development stage showing increase in fluorescent intensity of Dmd-citrine as development progresses. The error bars denote the variation among all the pixels at that particular stage. (l). Total pixel count of Dmd-citrine fluorescent plotted against developmental time showing that Dmd-citrine expression increase in spatial area over time. Scale bar = 20μm.</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452343, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g005", "stats"=>{"downloads"=>0, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dmd_Protein_expression_increases_both_in_intensity_and_area_over_development_/1452343", "title"=>"Dmd Protein expression increases both in intensity and area over development.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120396"], "description"=>"<p>(a). Single optical section confocal images of live 30hpf <i>ct90aGT</i> embryo show that Dmd-citrine fusion protein is localized at the somite border. Dmd is in green (citrine), nuclei are in blue (H2B-cerulean), and membranes are in red (membrane-mCherry mRNA injected). (b). Antibody staining for Citrine and endogenous Dmd in the trap line confirms their co-localization at the somite border. Anti-Citrine label is in green and anti-Dmd label is in red. (c-f). Antibody staining for Citrine and endogenous muscle proteins. Myosin heavy chain (c) and Tropomyosin (d) are expressed in the muscle fibers while Laminin (e) localizes to the extracellular matrix of the myosepta. Scale bar = 10μm (a), 20μm (b).</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452312, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g002", "stats"=>{"downloads"=>1, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_Gt_dmd_citrine_ct90a_trap_line_recapitulates_endogenous_Dmd_protein_expression_/1452312", "title"=>"The <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> trap line recapitulates endogenous Dmd protein expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120413"], "description"=>"<p>(a, f, k) Schematic of <i>in situ</i> HCR experiment showing <i>dmd</i> and <i>citrine</i> HCR probe binding sites within <i>dmd</i> and <i>dmd-citrine</i> transcript. The <i>dmd</i> HCR probe should bind to both <i>dmd</i> and <i>dmd-citrine</i> transcripts, while <i>citrine</i> HCR probe binds only to <i>dmd-citrine</i> transcript. (b-e, g-j, l-o). <i>in situ</i> HCR analysis of <i>dmd</i> (blue) and <i>dmd-citrine</i> (green) transcript in wild type (b-e), heterozygous (g-j) and homozygous (l-o) <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> embryos. <i>Tmp3</i> transcripts counter-stain muscles in red. Insets in (d),(i) and (n) show zoomed in view of transcription sites in the nuclei. Two dots are detected in homozygous embryos (k-n) whereas only one dot is detected in the nuclei of heterozygous <i>ct90aGT</i> embryos (f-i) while no citrine dots are detected in wild types embryos (a-d), consistent with the copy number of citrine inserted in the respective embryos. (e, j, o) Magnified images of the three types of embryos with nuclei stained in DAPI and the transcripts indicated by arrowheads. Scale bar (b-d, g-i, l-n)20μm (e,j,o)10μm.</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452324, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g003", "stats"=>{"downloads"=>1, "page_views"=>37, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_Gt_dmd_citrine_ct90a_trap_line_enables_visualizing_differential_expression_in_homozygous_and_heterozygous_embryos_via_in_situ_HCR_/1452324", "title"=>"The <i>Gt(dmd-citrine)</i><sup><i>ct90a</i></sup> trap line enables visualizing differential expression in homozygous and heterozygous embryos via in situ HCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120480", "https://ndownloader.figshare.com/files/2120481", "https://ndownloader.figshare.com/files/2120482", "https://ndownloader.figshare.com/files/2120483", "https://ndownloader.figshare.com/files/2120484"], "description"=>"<div><p>Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, <i>Gt(dmd-citrine)<sup> ct90a</sup></i>). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to <i>dmd</i> mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.</p></div>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452385, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0128944.s001", "https://dx.doi.org/10.1371/journal.pone.0128944.s002", "https://dx.doi.org/10.1371/journal.pone.0128944.s003", "https://dx.doi.org/10.1371/journal.pone.0128944.s004", "https://dx.doi.org/10.1371/journal.pone.0128944.s005"], "stats"=>{"downloads"=>2, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Spatio_Temporal_Differences_in_Dystrophin_Dynamics_at_mRNA_and_Protein_Levels_Revealed_by_a_Novel_FlipTrap_Line_/1452385", "title"=>"Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-06-17 04:17:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/2120427"], "description"=>"<p>(a-d). Chromogenic <i>in situ</i> hybridization for <i>dmd</i> in wild type embryos at 18hpf (a,b) and 22hpf (c,d). (a,c) Wide field image of 18hpf and 22hpf embryos stained for <i>dmd</i> expression. (b,d) 3D projections of confocal z-stack of embryos in (a) and (c), respectively. Fluorescent signal of NBT/BCIP stain for <i>dmd</i> transcript reveals spatial confinement of transcript between somites. (e-p). Confocal image of <i>dmd</i>-<i>citrine</i> transcript (f,j,n) detected by <i>in situ</i> HCR and antibody staining for DMD-citrine protein (e,i,m) in <i>Gt(dmd-citrine)</i><sup>ct90a</sup> (e-l) and wildtype (m-p) embryos at 18hpf and 26hpf. Counter-stain with antibody to Ctnnb1 in blue (g,k,o). (h,l,p) Merged of image of (e-g, i-k, and m-o). Comparison of protein and transcript expression at 18hpf (e-h) and 26 hpf (i-l) in <i>Gt(dmd-citrine)</i><sup>ct90a</sup> embryos show that DMD protein localize exclusively to the myosepta while <i>dmd-citrine</i> mRNA is expressed in the cytoplasm at 18hpf and becomes more localized to the myosepta at 26hpf. Dmd-citrine expression in the nucleus can be seen as dots that appear similar in distribution between 18hpf and 26hpf. (m-p) DMD-citrine protein (m) and transcript (n) are not detected in wildtype embryos. Anti-Citrine antibody staining is in green, <i>citrine</i> mRNA HCR is in red and anti-Ctnnb1 antibody is in blue. Scale bars (a) 50μm (b,m) 20 μm.</p>", "links"=>[], "tags"=>["Dmd protein localization", "Novel FlipTrap Line Dystrophin", "dmd transcript expression", "zebrafish gene trap line", "myosepta", "dmd mRNA localization", "Dmd protein", "Dmd protein localizes", "Protein Levels Revealed"], "article_id"=>1452338, "categories"=>["Biological Sciences"], "users"=>["Frederique Ruf-zamojski", "Vikas Trivedi", "Scott E. Fraser", "Le A. Trinh"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128944.g004", "stats"=>{"downloads"=>0, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_dmd_mRNA_and_Dmd_protein_expression_show_differences_in_spatial_expression_/1452338", "title"=>"<i>dmd</i> mRNA and Dmd protein expression show differences in spatial expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-06-17 04:17:17"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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