Spatial temporal coverage and stability of Multibow labeling.

<p><b>a.</b> Spatial and cell type coverage of Multibow. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The whole 4dpf larva was imaged in 2 channels (G/R). Positive cells can be seen distributed from head to tail throughout the larva, indicating high spatial coverage. In inserts 1 and 2, distinctly shaped skin, muscle, mesenchymal and neural cells can be observed by cytoplasmic or membrane Multibow labeling. Scale bars: 100μm. <b>b.</b> Temporal stability of labeling. The embryo was injected with 6 Multibow colors (mR/mG/nR/nG/R/G) at single cell stage and heat-shocked at 1 day-post-fertilization (dpf) for 2 hours. The same embryo was imaged once per day to 11dpf. The persistence of labeling indicates genomic insertion of Multibow cassettes. Red patches around the eye and along the gut are auto-fluorescence. Enlarged views of white boxed areas show that the area is stably fluorescent. Scale bar in enlarged views: 100μm. <b>c.</b> Label stability of color codes over time. The embryo was injected with 12 (B/G/Y/R) Multibow constructs at one cell stage. Heat-shock of this tg(<i>hsp70</i>:<i>cerulean-cre</i>) individual was at 30hpf (duration: 2 hours). Its developing larval tail fin was imaged every 24 hours starting at 54hpf using four channels (B/G/Y/R). The color codes of the cells remain unchanged despite fluorescent intensity differences at different days, allowing identification of the same cells/clones(e.g., α and β, shown in enlarged regions marked by white boxes). Color codes: α: nG/nY; β: mB. Scale bar: 100μm. See also Fig d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s002" target="_blank">S2 Fig</a>.</p

Design and test of Multibow in zebrafish.

<p><b>a.</b> Modified “Brainbow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.ref001" target="_blank">1</a>]” cassette that allows a binary ON/OFF switch. <b>b.</b> Multibow Strategy. Each cell harbors multiple different ON/OFF cassettes to generate random color “digital” barcodes upon Cre-mediated recombination. <b>c.</b> Table of Multibow Tags and Fluorescent Proteins (FPs). <b>d.</b> Diversity of color codes. Image is a densely labeled region along the trunk of a 40hpf <i>hsp70</i>:<i>cerulean-cre</i> embryo injected with all 21 Multibow constructs and heat-shocked at 10hpf for 1 hour. The color and tag diversity generates barcodes for cell clones that appear random and diverse. Intensity differences further help distinguish cells from neighbors visually. The Composite image is made from the green, yellow (turned to blue) and red panels. 3 different clones are highlighted by α, β, γ and corresponding arrows. Scale bar: 10μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127822#pone.0127822.s006" target="_blank">S3 Table</a>. <b>e.</b> Partial table of clones of different color codes found in <b>d.</b>. The colored square labels of the top row indicate nuclear, membrane and cytoplasmic, respectively. A black square in the table indicates this clone being positive for the corresponding color. Distinct "barcodes" form for different clones. The α, β, γ clones are indicated by arrows. The number of annotated cells labels (~30) represents a large fraction of cells found in the image in <b>d</b>, which contains ~50 cells. The fact that most of these cells have a color code distinct from any other cell (except clones that have the same color) show that Multibow label is highly random.</p

Examples of Multibow Cell Tracing in Development and Regeneration.

<p><b>a.</b> Cranial facial development mapped by Multibow. The embryo was heat-shocked at 6hpf. 4 channels (B/G/Y/R) were used. The left face of the larva was imaged. Red boxes: regions highlighted in <b>b.</b> and <b>c.</b>. Scale bars: 50μm. <b>b.</b> Lineage relationship between neuromast hair cells. Dashed line circle indicates the hair bundle. Multibow labeled hair cell color codes: 1(mB/nY/R), 2(mB/mG/nR), 3(mB), 4(nG), 5(mB/nR), 6(R). The same pattern was already observed at 30hpf. Scale bars: 10μm. <b>c.</b> Identification of cells that undergo remarkable morphological changes during semicircular canal formation. Arrows: initial locations of the two mesenchymal cells that span the projection later. Grey circle: posterior otolith. Scale bars: 50μm. <b>d.</b> Clonal expansion near the eye over long time periods. The embryo was injected with 12 constructs (B/G/Y/R) and heat-shocked at 10hpf. Arrows indicate locations of identified clones α (nG), β (nG/R), γ (nY/mR). These clones can be seen amplified in number at 54hpf or 129hpf (α: 2 to 4; β: 2 to 4; γ: 2 to 3). Scale bar: 100μm. <b>e.</b> Multibow analysis of regeneration in the larval tail. Heat-shock labeling (1 hour), amputation and imaging were performed as labeled in the timeline. Immediately after amputation, the tissue shrank and cells near the wound converged (the images overlay may appear to be slightly out of register due to the changes of the live tissue during the acquisition of different channels, cell identification is not affected as these changes are small and predictable). By 2 days after amputation, most cells that had converged at the frontier of the wound were gone (their unique color codes disappeared, red arrowheads). The regenerated tissue came from clonal expansion of cells away from the frontier (highlighted examples in enlarged view from the white boxes 1 and 2). These clones show lineage restriction to the original cell type (the morphology of cells in the same clone remains similar, e.g., the blue cells in box 1 increased in number while size and shape do not have major changes.). Scale bars: 50μm.</p

Inhibition of c-Myc blocked proliferation and down-regulated vimentin during HC regeneration.

<p>(A) A significant reduction in the number of proliferating neuromast cells (BrdU⁺) was seen 18 or 24 hrs after c-MYC inhibitor Int-H1-S6A, F8A (Myc-pep, 100 nM) treatment. Ctr, time-matched control fish without neomycin treatment. (B) c-MYC peptide inhibitor mainly blocked proliferation-derived HCs (HC-BrdU⁺) at 72 hrs. ***<i>p<0</i>.<i>001; **p<0</i>.<i>01</i>. A and B, n = 15 for each group. Two independent experiments were performed with similar results. (C-H) <i>In situ</i> hybridization of <i>atoh1a</i> on 5-dpf zebrafish neuromasts 18 (C-E) or 24 hrs (F-H) after neomycin treatment, with either Myc-pep or DMSO in the media. Ctr, time-matched control fish without neomycin treatment. <i>atoh1a</i> up-regulation by neomycin treatment was blocked in the Myc-pep treatment group (D-E, G-H). (I-Q) Vimentin was down-regulated by c-Myc during HC regeneration. 5-dpf neuromasts were labeled with vimentin and DAPI 18 hrs after neomycin treatment, with either Myc-pep (O-Q) or DMSO (L-N) in the media. No Trt, time-matched control fish without neomycin treatment (I-K). Scale bars: 10 μm.</p

Myc inhibition suppresses HC regeneration in zebrafish neuromasts.

<p>(A) HCS1-labeled HC number was significantly reduced after treatment by c-MYC inhibitor 10058F4 that was dose-dependent. ***<i>p<0</i>.<i>001</i>. n = 20 larvae for each group. (B) A significant reduction in HC number was observed after the treatment by Int-H1-S6A, F8A, a peptide inhibitor of c-MYC, which was dose-dependent. ***<i>p<0</i>.<i>001</i>. n = 15 larvae for each group. Three independent experiments were performed for each comparison study with similar results. (C-H) Inhibitor Int-H1-S6A, F8A blocked the expression of <i>c-myc</i> target gene <i>odc1</i>. <i>In situ</i> hybridization showed up-regulation of <i>odc1</i> expression in the neuromast 3 or 6 hrs after neomycin treatment (D, G); whereas Int-H1-S6A, F8A markedly suppressed <i>odc1</i> expression (E, H). Scale bars: 10 μm.</p

Inhibition of Fgf signaling suppresses HC regeneration.

<p>(A) A significant reduction in the number of HCs (HCS1⁺) regenerated after SU5402 treatment that was dose-dependent. Ctr, time-matched fish without neomycin treatment. (B) A significant reduction in the number of HCs regenerated in <i>hsp70l</i>:<i>dn-fgfr1</i>:<i>GFP</i> (Hsp) zebrafish neuromasts at 32°C or 37°C. AB: wild type AB fish. Ctr, time-matched transgenic fish without neomycin treatment. (C) A significant reduction in proliferation and transdifferentiation derived HCs after 20 μM SU5402 treatment for 72 hrs. ***<i>p<0</i>.<i>001; **p<0</i>.<i>01</i>. For all statistical analysis, n = 15 for each group. Three (for A) and two (for B,C) independent experiments were performed with similar results. (D) Fgf targets <i>etv4</i> and <i>spry4</i> were down-regulated by SU5402 (20 μM) in L1 neuromasts during HC regeneration. Scale bars: 10 μm.</p

Neuromast SCs with different capacities in HC regeneration and proliferation.

<p>Hybrid larvae of <i>pou4f3</i>:<i>GFP</i> and <i>ET20</i> fish ablated in HCs only (A,D), HC/<i>fgfr1a</i>(+) SCs (B,E), or HC/<i>fgfr1a</i>(-) SCs (C,F) were stained with HCS1 (A-C) or BrdU (D-F) antibody, to illustrate proliferating (BrdU⁺) and regenerated HCs (HCS1⁺). (G) Quantification showed that only HC/<i>fgfr1a</i>(+) SC ablation significantly reduced the regenerated HCs. (H) Quantification showed that HC/<i>fgfr1a</i>(+) SC ablation increased the number of proliferating cells; whereas HC/<i>fgfr1a</i>(-) SC ablation reduced the proliferating cells. (G,H) ***<i>p<0</i>.<i>001; *p<0</i>.<i>05</i>. n = 12 in each group. Scale bars: 10 μm.</p

Expression of Myc and Fgf pathway genes during HC regeneration in zebrafish neuromasts by <i>in situ</i> hybridization.

<p><i>mycb</i> (A-D), <i>fgf10a</i> (E-H), <i>fgf3</i> (I-L), <i>fgfr1a</i> (M-O), <i>fgfr2</i> (P-R) expression were shown in the lateral line neuromast L1 from 5-dpf zebrafish at different time points following neomycin treatment (e.g. Neomycin-6hr, 6 hrs after neomycin treatment). Ctr, untreated fish larvae. Dotted blue lines marked the boundary of neuromasts. Bold dotted lines marked the expression areas (M,O,P,R). (S) An illustration of a neuromast demarcated by differential <i>fgfr</i> expression patterns, including HC region, <i>fgfr1a(+)</i> only region, <i>fgfr1a/r2(+)</i> region and <i>fgfr1a(-)</i> region. A-P, anterior-posterior; D-V, dorsal-ventral. Scale bars: 10 μm.</p