KA″ specification depends on Hh signaling.

<p>(A) Quantification of the total number of KA″ cells at 24 hpf under different conditions. Cyclopamine (Cyc) was treated from indicated stage until 24 hpf. Embryos injected with dnPKA or Shh mRNA showed increase in total number of KA″ cells compared to uninjected control embryos. (B) Cyclopamine-treated embryos (from 8 hpf to 24 hpf) abolished <i>tal2</i> expression in the LFP domain, while overexpression of dnPKA and Shh induced ectopic <i>tal2⁺</i> cells. Note that the few <i>tal2⁺</i> cells in cyclopamine-treated embryos are located dorsal to the LFP domain. Images shown are lateral views of embryos at 24 hpf. The dorsal-ventral extent of the LFP domain is indicated by brackets. Scale bar: 50 µm.</p

Temporal profiles of Hh response visualized by a <i>Ptc1-Kaede</i> reporter.

<p>(A) Schematic drawing of the <i>Ptc1-Kaede</i> BAC reporter. A cassette containing Kaede and Kanamycin resistant gene was recombined to replace the first exon of <i>ptc1</i>. (B) <i>Ptc1-Kaede</i> fish showed <i>kaede</i> expression in a pattern similar to the expression of <i>ptc1</i> in wild type embryos at 19-som stage. (C) Inhibition of Hh signaling using cyclopamine blocked <i>Ptc1-Kaede</i> expression, while overexpression of dnPKA mRNA induced ectopic expression of the transgene. Fluorescent signal in cyclopamine-treated fish is due to auto-fluorescence of the yolk (asterisk). (D) Schematic drawings of PHRESH analysis. Photoconversion of the <i>Ptc1-Kaede</i> reporter can be used to determine the timing of Hh response (see text for details). (E) <i>Ptc1-Kaede</i> fish were photoconverted at 25 hpf, and imaged at 36 hpf. Single optical sections of a lateral view, dorsal view, and cross-section are shown. Arrows indicate <i>Ptc1-Kaede^red</i> cells in the lateral floor domain. Note that dorsally located KA′ cells also only express <i>Ptc1-Kaede^red</i> (arrowheads). (F) <i>Ptc1-Kaede</i> fish were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 35 hpf. Arrows indicate GABA-positive KA″ cells. (G) <i>Gata2-GFP</i> fish were co-labeled with <i>ptc1</i> (green), and the GFP antibody (red). Images shown are the dorsal view of an 18 hpf embryo (top) and the lateral view of a 24 hpf embryo (bottom). KA″ cells are indicated by arrows. Scale bars: 200 µm in B–C and 20 µm in E–G.</p

Lineage analysis of the LFP domain.

<p>(A) Schematic drawings of scatter labeling and time-lapse imaging. <i>Gata2-GFP</i> fish were injected with <i>H2B-mCherry</i> mRNA (red) into a single blastomere at 16- to 32-cell stage. Scatter labeled embryos with nuclear mCherry expression (red) were imaged in the dorsal view starting at 3-somite stage (11 hpf) for about 11 hours. At the end of the time-lapse, an image with both the green and red channels was acquired to identify <i>Gata2-GFP</i>-expressing KA″ cells (green). (B) Observed division patterns in the LFP domain. Of a total of 25 cell divisions tracked, 16 were LFP/LFP divisions, 6 were KA″/LFP divisions, and 3 were KA″/KA″ divisions. KA″ cells (green) were identified base on the expression of <i>Gata2-GFP</i> reporter. (C) <i>Gata2-GFP</i> fish (green) was scatter labeled by <i>H2B-mCherry</i> (red) and imaged from 11 hpf for about 11 hours. Two examples are shown. Top panel: merged images with both the red channel and the bright field of a single optical slice at the start of the movie at 11 hpf. The underlying notochord (brackets) is visible but out of focus. Middle and bottom panels: the merged image with both green and red channels and the green channel alone of a confocal projection at the end of the movie around 22 hpf. KA″ cells (arrows) can be distinguished from LFP cells (arrowheads) based on the expression of <i>Gata2-GFP</i> (green) at 22 hpf. Medial floor plate cells are indicated by white dots. Lineage related cells confirmed by cell tracking are indicated (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002762#pgen.1002762.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002762#pgen.1002762.s004" target="_blank">Video S1</a>). For example, cell 1 generates a KA″ cell (cell 1a) and an LFP cell (cell 1b). Of the 7 examples shown here, cells 1 and 6 undergo KA″/LFP divisions, cell 7 undergoes KA″/KA″ division, and cells 2–5 divide symmetrically giving rise to two LFP cells (LFP/LFP divisions). Note that cells 4 and 6 are more dorsally located and therefore not in focus in images at 11 hpf (top panels). Scale bars: 20 µm.</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

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

Interaction between Notch and Hh signaling in KA″ specification.

<p>(A) Embryos treated with DMSO alone, compound E, cyclopamine, or both compound E and cyclopamine from 14 hpf to 25 hpf, were stained for the expression of <i>tal2</i>. (B) Quantification of total number of KA″ cells in experiments shown in A. Note that the fewer data points in compound E treatment is due to the fact that most compound E-treated embryos had many <i>tal2</i>-positive cells in close clusters which prevents reliable scoring. The two data points thus underestimate the total number of KA″ cells in E-treated embryos. Cyc: cyclopamine. (C) <i>hsp-Gal4; UAS-NICD</i> embryos and non-transgenic sibling controls were left uninjected or injected with Shh mRNA, heat-shocked at 11.5 hpf, and stained at 24 hpf for the expression of <i>tal2</i>. Brackets in A and C indicate the dorsal-ventral extent of the LFP domain. Scale bars: 50 µm. (D) <i>hsp-Gli1</i> embryos and non-transgenic controls were injected with compound E at 15 hpf, and heat-shocked at 16 hpf, and stained at 22 hpf for the expression of <i>nkx2.9</i>. Arrows denote the two rows of LFP domains in dorsal views. (E) Model of KA″ specification. At the early stage (t₀), LFP progenitors have high level of Notch signaling activity and thereby maintain the progenitor state and Hh responsiveness (Notch^ON Hh^ON). Active Hh signaling in progenitor cells is required for specifying the KA″ identity in subsequent cell divisions. LFP progenitors can undergo three different types of divisions: symmetric LFP/LFP divisions, asymmetric KA″/LFP divisions (shown here), and symmetric KA″/KA″ divisions. Divisions of LFP progenitors at t₁ generate daughter cells with similar competence to either acquire the KA″ fate or maintain the LFP progenitor fate. Cell-cell interactions or stochastic fluctuations in Notch signaling result in cells with different levels of Notch signaling (t₂). Cells that maintain high levels of Notch signaling will remain as LFP progenitors and continue to respond Shh (Notch^ON Hh^ON). In contrast, cells that have attenuated Notch signaling will lose Hh response and differentiate into KA″ interneurons (Notch^OFF Hh^OFF). Since sustained Notch or Hh signaling disrupt the differentiation of KA″ cells, formation of KA″ cells initially depends on the activation and then the attenuation of Notch and Hh signaling.</p

Prolonged Hh response interferes with KA″ specification.

<p>(A) Quantification of KA″ specification in embryos overexpressing GFP-Gli1. <i>hsp-GFP-Gli1</i> embryos and their non-transgenic sibling controls were heat-shocked at indicated stages, and stained at 25 hpf for the expression of <i>tal2</i>. (B) <i>hsp-GFP-Gli1</i> and control embryos were heat-shocked at 14 hpf, and stained at 24 hpf for the expression of <i>tal2</i> and <i>nkx2.9</i>. Induction of GFP-Gli1 results in a reduction of <i>tal2</i>-positive KA″ cells (arrows) and expansion of <i>nkx2.9</i>-expressing LFPs. (C) <i>Ptc1-Kaede</i> control embryos, and <i>Ptc1-Kaede; hsp-GFP-Gli1</i> embryos were heat-shocked at 14 hpf, photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Induced expression of GFP-Gli1 results in a reduction of KA″ cells (arrows). Note that at 36 hpf, GFP-Gli1 expression has minimal contribution to the green fluorescence. (D) <i>Ptc1-Kaede</i> control embryos, and <i>Ptc1-Kaede</i> embryos injected with Shh mRNA, Ptc1 and Ptc2 morpholinos, or dnPKA mRNA were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Arrows indicated GABA-positive cells in the LFP domain. Note that Shh and dnPKA overexpression induced many ectopic GABAergic neurons (arrowheads) throughout the dorsal-ventral axis of the spinal cord, and most of them appeared to lose Hh response by 24 hpf as indicated by the expression of <i>Ptc1-Kaede^red</i>. Scale bars: 20 µm.</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

Notch signaling in KA″ specification.

<p>(A) Wild type embryos at 18 hpf were double labeled with <i>tal2</i> (red) and <i>hes5</i>, <i>her12</i>, <i>her2</i>, or <i>her4</i> (green). <i>tal2</i>-expressing KA″ cells (arrows) lack expression of <i>hes5</i>, <i>her12</i>, <i>her2</i>, or <i>her4</i>. Note that <i>hes5</i>, <i>her12</i>, <i>her2</i>, and <i>her4</i> are expressed in only a subset of LFP cells, which might reflect the oscillating nature of these genes in neural progenitor cells as previously reported <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002762#pgen.1002762-Shimojo1" target="_blank">[49]</a>. Dorsal views are shown. (B) <i>hsp-Gal4; UAS-NICD</i> embryos and non-transgenic control embryos were heat-shocked at 10 hpf, and stained at 25 hpf for the expression of <i>tal2</i> and <i>nkx2.9</i> (top panel). Embryos were treated with DMSO or compound E at 13 hpf, and stained at 25 hpf for the expression of <i>tal2</i> and <i>nkx2.9</i> (bottom panel). <i>tal2</i> and <i>nkx2.9</i> staining is shown in lateral and dorsal views, respectively. Brackets indicate the extent of the LFP domain in lateral views, and arrows denote the two rows of LFP domains in dorsal views. (C) Quantification of KA″ specification under different conditions. KA″ cells were scored by the expression of <i>tal2</i> at 25 hpf. Note that the fewer data points in compound E treatment at 13 hpf is due to the fact that most compound E-treated embryos had many <i>tal2</i>-positive cells in close clusters which prevents reliable scoring. Data shown therefore underestimate the total number of KA″ cells under this condition. (D) <i>Ptc1-Kaede</i> embryos were treated with DMSO or compound E at 14 hpf, photoconverted at 25 hpf, and stained with the GABA antibody (blue) at 37 hpf. Compound E treatment induced ectopic GABA-positive cells in the LFP domain (arrows), and abolished the expression of <i>Ptc1-Kaede^green</i> (middle panel). Scale bars: 20 µm in A, D and 50 µm in B.</p

Geometric structure classification based on eigen-system.

<p>An overview of the local intensity structures determined by their eigen-system. Parameters <b>A</b>, <b>B</b>, and <b>S</b> refer to individual terms in the planarity filter (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi.1002780.e055" target="_blank">Equation 1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002780#pcbi.1002780.e056" target="_blank">2</a>). These terms are specified as ratios of individual eigenvalues to enhance the identification of planes relative to rods and ball structure classes.</p