Iridophore enriched genes.

<p>RPKM values for genes expressed in iridophores at least 30-fold greater than melanocytes and RPE, and 100-fold greater than embryos.</p

Candidate control genes are differentially expressed.

<p>Selected genes indicative of pigment cell identity or shared functions are shown.</p

The guanine synthesis cycle is highly enriched in iridophores.

<p>Shown is a model for guanine production based on transcriptome data as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067801#pone-0067801-t006" target="_blank">Table 6</a>. Genes that are statistically enriched compared to melanocytes are shown in bold, those not statistically different are in normal font. The arrow thicknesses correspond to the fold changes in iridophores relative to whole embryos. Chemical structures are from the KEGG Compound database.</p

Shared gene expression among melanocyte and RPE.

<p>RPKM values for genes co-expressed in melanocyte and RPE at least 10-fold greater than iridophores and whole embryos, with a minimum of 10 RPKM.</p

Shared gene expression among melanocyte, RPE, and iridophore.

<p>Shown are RPKM values for genes co-enriched among the three pigment cell types at a level 100-fold greater than whole embryos, within a 2-fold change of each other, with a minimum RPKM of 4.</p

Shared gene expression among melanocytes and iridophores.

<p>RPKM values for genes expressed in melanocytes and iridophores at least 5-fold greater than RPE and whole embryos.</p

Purification procedure for melanocytes, iridophores, and retinal pigmented epithelium.

<p>Zebrafish are grown to the desired time point; shown in (A) is a six day old fish. (B) Fish are dissociated to a single cell suspension; black melanocytes (arrows) and reflective iridophores (arrowheads) are visible as a small percentage of all cells. (C) Cells are placed atop a Percoll density gradient and centrifuged. (D) The resulting cell pellet is resuspended and analyzed by FACS. Shown is a characteristic FACS plot demonstrating the relative positions of melanocyte and iridophore gates (ovals). Sorted iridophores are shown on the upper left of (D) under incident light. Sorted melanocytes are on the lower right using trans-illumination.</p

Schematic of cDNA library preparation.

<p>PolyA-selected mRNA (in red) is reverse transcribed using a polyT primer tailed with a universal primer (A). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067801#pone.0067801.s010" target="_blank">Table S3</a> for primer sequences. MMLV reverse transcriptase adds cytosines to the 3′ end of the 1st strand cDNA (in black), allowing for template switching and addition of the 3′ universal primer (B). PCR amplification of the library is followed by RsaI and AluI enzymatic digestion of cDNAs (C), followed by the standard Illumina library preparation steps of end-repair, a single adenine addition, Y-adapter ligation (D), PCR enrichment, and size selection (mock gel shown in E with yellow box indicating area of gel removed for DNA extraction), prior to flowcell generation and sequencing.</p

Overexpression of <i>kcnk5b</i> is sufficient to cause fin overgrowth.

<p>(A) Construct used to create <i>kcnk5b</i>-expressing clones via Tol2 transgenesis. (B) Individual fish expressing <i>kcnk5b</i> (W169L) (left) or <i>kcnk5b</i> (wt) (right) in mosaic clones display localized fin and barbel overgrowth. (C–F) Overgrowth is associated with DsRed expression (in red) within mesenchymal cells. (C) Calcein staining labels bone tissue (in green) of an overgrown fin (DsRed; <i>kcnk5</i>(W169L) expressing clone). (D) Mesenchymal clones are associated with increased segment length in the fin compared to non-overgrown DsRed negative regions. (E) Fibroblast-like cells appear as DsRed positive cells within the fin rays (dotted line) that surround DsRed negative vasculature (arrows in E and F) which extend along the actinotrichia (fibrils within dotted lines in F) towards the distal end of the fin. (G) Overgrown barbels show DsRed signal within the mesenchyme (area within dotted line) but not in the vasculature (arrow). (H) Number of clones associated with overgrowth in different <i>kcnk5b</i> variants. (I) Proportion of different cell types labeled in overgrown tissues. (J) Electrophysiological recordings of the non-conductive <i>kcnk5b</i> (GFGAAA) mutant in oocytes. Squares: <i>kcnk5b</i> (wt), purple stars: <i>kcnk5b</i> (F241Y)+<i>kcnk5b</i> (wt), blue circles: <i>kcnk5b</i> (W169L)+<i>kcnk5b</i> (wt), green triangles: + <i>kcnk5b</i> (GFGAAA)+<i>kcnk5b</i> (wt). Current was normalized to the measurement of wt current at 60 mV. Inset: DsRed+ fibroblasts in fish injected with the non-conductive construct do not lead to fin overgrowth.</p

Cell proliferation is increased in <i>alf</i> mutants.

<p>(A) Sections of wild type and heterozygous <i>alf</i> fins. No significant difference in cell size is seen in the two groups. (B) Antibody staining against PCNA on paraffin sections of regenerating fins 4 days post amputation (dpa). Chart shows percentage of proliferating nuclei (PCNA) over total nuclei (Hoechst). N = 3–4 sections of 4 individual fish **: p-value<0.01.</p