brainbow reporter sequence  (Addgene inc)

Journal: The EMBO Journal

Article Title: Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

doi: 10.1038/s44318-024-00136-y

Figure Lengend Snippet: ( A ) The palmuscle-Multi and myofiber:iCre#1 transgenic constructs. ( B ) Schematic drawing of individual myofibers before and after Cre activation. Addition of tamoxifen (Tam) activates Cre recombinase, which acts on Brainbow-based cassettes to convert label-free myofibers into color-barcoded myofibers. ( C ) Whole-animal view of a live palmuscle-Multi zebrafish larva at 10 dpf. ( D ) Magnified view of the pectoral fin, craniofacial, and trunk myofibers. ( E ) Representative cross-sectional view of a myotome (left). Schematic outlines of color-barcoded myofibers are shown on the right. ( F ) Color space analysis of 2057 individual myofibers from 32 myotomes captured from a single palmuscle-Multi . About 50 distinct hues were detected in live animals upon Cre activation. ( G ) The palmuscle-Multi and myofiber:iCre#2 transgenic constructs. ( H ) Timeline of the treatment and tracking scheme. ( I ) Schematic drawing of the Tg(palmuscle-Multi; myofiber:iCre#2) larva before and after Cre activation. ( J ) Whole-animal view of the Tg(palmuscle-Multi; myofiber:iCre#2) larva without Dox and Tam treatment. ( K ) The pectoral fin, craniofacial and trunk region displayed multicolor myofibers upon treatment with Dox and Tam. n = number of animals ( J ). Stitched image ( C , D , J ). Scale bars, 200 µm ( C , J ); 100 µm ( D , K ). dpf, days post-fertilization. .

Article Snippet: A 2.2-kb promoter sequence upstream of the mylpfa gene (Ju et al, ) was used to generate following transgenic lines: Tg(mylpfa:palm-mTurquoise2)as69 , Tg(mylpfa:H2A-mCherry)as70 , and Tg(mylpfa:Brainbow1.0L)as71 , and Tg(mylpfa:H2A-mCherry-2A-GC3AI)as72 , and Tg(mylpfa:LifeAct-mScarlet)as76 , the Brainbow reporter sequence was a gift from Joshua Sanes (Addgene plasmid # 18721) (Livet et al, ).

Techniques: Transgenic Assay, Construct, Activation Assay

Journal: The EMBO Journal

Article Title: Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

doi: 10.1038/s44318-024-00136-y

Figure Lengend Snippet: ( A ) Timeline of the tracking scheme. ( B – D ) Long-term time-lapse imaging of the same myofibers in different anatomical regions, including pectoral fin myofibers ( B ), craniofacial myofibers ( C ), and trunk myofibers ( D ). White arrows highlight myofibers that “disappear” from later time points. ( E , F ) Quantitative changes in the tagged myofiber number. Of note, the analyses include only larvae with standard length more than 9 mm at 28 dpf. ( G ) Schematic drawing of zebrafish at larval, juvenile and adult stages, reflecting their relative size difference. ( H ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 14, 42, 70, and 180 dpf. White arrows point to remaining myofibers. White dashed line encircles the area containing fast muscle fibers (42, 70, and 180 dpf). F310 Ab stains fast myofibers. ( I , J ) Quantitative analysis of tagged myofiber number ( I ), and percent area occupied by the tagged myofibers ( J ). The tagged myofiber numbers from two to three consecutive cross-sections were counted and averaged for each individual. ( K ) Timeline of the tracking scheme. ( L ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 29, 42, and 70 dpf. White arrows point to remaining myofibers at 42 dpf. White dashed line encircles the area containing fast muscle fibers. F310 Ab stains fast myofibers. ( M , N ) Quantitative analysis of tagged myofiber number ( M ), and percent area occupied by the tagged myofibers ( N ). The tagged myofiber numbers from two to four consecutive cross-sections were counted and averaged for each individual. ( O ) Timeline of the treatment and tracking scheme. ( P , Q ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 29, 43 dpf, and 10 mpf showed no leaky Cre activity ( P ). A short pulse of Dox and Tam labeled most of the trunk fast myofibers ( Q ). White dashed line encircles the area containing fast muscle fibers. Number of animals examined—8, 6, 4 (29 dpf, 43 dpf, 10 mpf, P ); 8, 5, 3 (29 dpf, 43 dpf, 10 mpf, Q ). F310 Ab stains fast myofibers. ( R ) Schematic drawing of the palmuscle-Multi cassettes before and after Cre activation. qPCR primers were designed to detect transcripts from the recombined Brainbow cassettes, targeting the common sequences of mCherry, mYFP, and mCerulean. ( S ) Timeline of the sampling scheme. ( T , T’ ) RT-qPCR analysis of the whole-animal myofiber loss at 14, 28, 42 and 70 dpf ( T ), and an enlarged view of the 70 dpf data ( T’ ). Of note, three separate body compartments—Anterior (A), Middle (M), and Posterior (P)—were collected at 70 dpf, owing to the substantial size of the fish at this stage. Data from biological replicates are shown as mean ± standard deviation ( I , J , M , N ) or mean ± standard error ( T, T’ ). m = myofibers ( E , F ). n = number of animals ( E , F , I , J , M , N ) or biological repeats ( T , T’ ). Stitched image ( H , L , P , Q ). Scale bar, 100 µm ( B – D ); 100 µm ( H , 14 dpf) and ( L , P , Q , 29 dpf); 300 µm ( H , L , 42 dpf; P , Q , 43 dpf); 500 µm ( H , L , 70 and 180 dpf; P , Q , 10 mpf). dpf, days post-fertilization. mpf, months post-fertilization. .

Article Snippet: A 2.2-kb promoter sequence upstream of the mylpfa gene (Ju et al, ) was used to generate following transgenic lines: Tg(mylpfa:palm-mTurquoise2)as69 , Tg(mylpfa:H2A-mCherry)as70 , and Tg(mylpfa:Brainbow1.0L)as71 , and Tg(mylpfa:H2A-mCherry-2A-GC3AI)as72 , and Tg(mylpfa:LifeAct-mScarlet)as76 , the Brainbow reporter sequence was a gift from Joshua Sanes (Addgene plasmid # 18721) (Livet et al, ).

Techniques: Imaging, Activity Assay, Labeling, Activation Assay, Sampling, Quantitative RT-PCR, Standard Deviation