Additional file 7: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Code used to produce the plot in Figs. 4 and 5, Additional file 3: Figure S1. (TXT 14 kb

Additional file 5: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Results from KASP genotyping used for Fig. 4d. (TXT 910 bytes

Additional file 2: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Tutorial for the bioinformatics including sgRNA/primer deisgn and MiSeq analysis. (PDF 7778 kb

Additional file 4: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Amplicon screening data used for Fig. 4a-c. (TXT 142 kb

Additional file 3: Figure S1 and Figure S2. of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Comparison of induced indels between germline and somatic tissues. (a) Plot shows the same data as Fig. 4a, split into separate plots for each sgRNA. (b) Plot of frequencies for individual variants in sperm versus fin clip (same data as in Fig. 4b with full axes). (PDF 374 kb

Clonal analysis of laser-induced myocellular injuries.

<p>(A–B) Laser-injury and recovery within the same embryo. (A) Left: DIC image superimposed with image of individual muscle cells marked by Tg[<i>βactin:α-actinin-gfp</i>] expression (false colored in yellow). Right: laser-injury was performed by targeting one of the α-Actinin-GFP positive muscle cells. Immediately upon laser-injury, α-Actinin-GFP expression within the damaged cell is diminished (arrowheads). Scale bar: 50 µm. (B) Confocal images of two z-scan planes of an immunohistochemical staining show strong Xirp1 expression within 2.5 hours after laser injury in muscle cells directly adjacent to the targeted cell that was most severely affected by the laser-injury (arrowheads). Pictures on the right are details from the insert (white box on the bottom left picture). Green: <i>βactin</i>:α-Actinin-GFP; red: Actin; blue: Xirp1. Scale bar: 50 µm. (C) Confocal z-scan projection of an immunohistochemical staining 5 hours after laser-induced injury reveals that strong Xirp1 expression correlates with a pattern of Tropomyosin distribution that is different from unaffected regions of the somite. Green: Tropomyosin; red: Actin; blue: Xirp1. Scale bar: 20 µm.</p

Characterization of the P47 antibody.

<p>(A) Myotendinous junction labeling by the P47 antibody which recognizes Xirp1α/γ is absent in <i>xirp1^sa0046</i> mutant embryos at 24 hpf. This staining was performed upon mild fixation. (B) Clonally expressed Xirp1γ is sensitively detected upon standard fixation conditions by the P47 antibody within somitic muscle tissue. Truncated Xirp1γ^ΔFilCBD-GFP which lacks the Filamin C binding domain including the P47 epitope is not detected by the antibody. Under standard fixation conditions, Xirp1α/γ is not detected at the myotendinous junctions. Green: GFP or Xirp1γ-GFP fusion protein; red: Actin; blue: Xirp1α/γ. All scale bars: 25 µm.</p

Transcriptome analysis for genes regulated by Galanthamine treatment in zebrafish.

<p>Summary table listing those genes that are most strongly upregulated in the acetylcholinesterase inhibitor Galanthamine-induced model of muscle injury. For treatment, zebrafish embryos were continuously incubated with Galanthamine between the end of gastrulation and several days of development. Comparisons of fold changes (FC) at 56 hpf and 72 hpf are shown for those genes showing the strongest expression changes. <i>xirp1</i> is more than three-fold upregulated at both time-points under conditions of myocellular injury.</p

Expression and localization of Xirps within Galanthamine- and laser-induced myocellular wounds.

<p>(A) Schematic diagram summarizing the temporal order of Xirp expression within somitic muscle and in myocellular wounding assays. (B) Galanthamine (GAL) treatment between 80% epiboly and 2 dpf causes severe disruptions of somitic muscle organization and myofibrillar disarray (red: Actin) in 2 dpf zebrafish embryos. Notably, Xirp1 (green) is strongly expressed and localizes within cells most strongly disrupted by the treatment. These effects are completely reversible within several hours of recovery. Scale bar: 50 µm. (C) Details from inserts indicated in B (green: Xirp1; red: Actin). Scale bar: 10 µm. (D, E) Similarly, laser-induced muscle injury induces ectopic Xirp1 and Xirp2a localization to damaged myofibrils. In comparison, Xirp2b is not yet expressed at 33 hpf. Green: Xirp1, Xirp2a or Xirp2b; red: Actin. Arrows indicate the position of laser-induced injury within somitic tissue. Scale bars: 10 µm. Embryos were injured at 27 hpf (D) or 29 hpf (E) and fixed at 33 hpf (D) or 31.5 hpf (E), respectively.</p

Rapid <i>xirp1</i> transcriptional response to myocellular injury.

<p>Whole-mount <i>in situ</i> hybridizations on 33 hpf embryos which were laser-injured either at 29 hpf (bottom row) or at 32 hpf (middle row) at the level of three different somites (dotted lines). <i>xirp1</i> mRNA transcriptional response occurs within one hour after injury and is no longer detectable at 3.5 hours after injury in WT. There is a lack of <i>xirp1</i> mRNA expression upon laser-induced myocellular injury in <i>xirp1^sa0046</i> mutants. Scale bar: 100 µm.</p