Analysis of differential dorsal-ventral <i>asip1</i> gene expression.

<p>Asip1 was differentially expressed in ventral non-pigmented skin or dorsal pigmented skin in turbot (A) and sole (B). <i>Asip1</i> gene expression was quantified by absolute qRT-PCR. The average <i>asip1</i> gene copy number per µg of primed cDNA was calculated from 5 individuals analyzed each time in triplicate. Data are expressed as mean ± SEM. Comparisons of numerical data were made by paired Student t-tests. *P<0.05.</p

Views of dorsal skin of turbot injected and eletroporated <i>in vivo</i> to evaluate the effect of asip 1 overexpression on skin paling.

<p>Animals were injected with about 10 µg of capped sense (B) or antisense (D) mRNA per cm² of dorsal skin and the effect was evaluated 4 days post injection/electroporation. Dorsal skin of control non-treated turbots are shown in A, C, E, and G. E and F show dorsal skin of control (E) and injected/electroporated animals with capped sense eGFP-mRNA (F) animals under fluorescent incident light w. G and H display animals shown in E and F under brilliant incident light. . Fluorescence was determined with a binocular Leica Stereoscope M165FC with digital camera (Leica Microsystem). Images were processed with Photoshop 7.0 (Adobe Systems) programs. Dorsal views, anterior to the right. Scale bars: 0.6 cm.</p

Phylogenetic tree of asip and agrp amino acid sequences built using CulstalX, which uses the Neighbor-Joining method on a matrix of distances.

<p>Numbers at branch nodes represent the confidence level of 1000 bootstrap replications. Phylogenetic analysis were done also by maximum likelihood using Seaview free software and no considerable differences were found.</p

RT-PCR analysis of the tissue specific expression pattern of asip1.

<p>(A) Turbot and (B) sole.</p

Analysis of <i>asip1</i> gene expression in pseudoalbino flatfish.

<p>Pseudo-albinism phenotype present in cultured turbot (A) and sole (C). <i>Asip1</i> was differentially expressed in non-pigmented white or pigmented brown dorsal skin areas in turbot (B) and sole (D). <i>Asip1</i> gene expression was quantified by absolute qRT-PCR. The average <i>asip1</i> gene copy number per µg of primed cDNA was calculated from 5 individuals analyzed each time in triplicate. Data are expressed as mean ± SEM. Comparisons of numerical data were made by paired Student t-tests. *P<0.05. Scale bars: 1 cm.</p

Expression of <i>asip 1</i> gene during early development.

<p>RT-PCR analysis of the temporal expression pattern of <i>a</i>sip1 in turbot (A) and sole (B). Hours post-fertilization, hpf; days post-fertilization, dpf.</p

Alignment of agouti-signaling protein (asip) and agouti-related protein (agrp) amino acid sequences.

<p>Dashes were introduced to improve alignment. Orange boxes indicate the last residue of the predicted signal peptide. Black boxes show amino acid residues conserved in all sequences. Green boxes show residues only conserved in asip1 sequences. Red boxes indicate residues only conserved in agrp1 sequences. Yellow boxes indicate basic residues before cysteine domain. Blue boxes show residues of the short tail present in all agrp sequences. Purple boxes indicate putative glycosilation sites. Lines joining cysteine residues indicate putative disulfide bonds forming the cysteine domain. Arrow shows conserved motif for agrp post-transcriptional processing.</p

Panel A shows the effects of oral T3 (500 μg/g of food) on zebrafish pigment phenotype.

<p>Fish were feed with control (CTRL) or T3-supplemented (T3) diet during 15 days and then fed with CTRL diet for 15 additional days (T3/CTRL). Higher magnification of melanic stripes in CTRL (B) and T3 (C) fish. D1 and D2, dorsal stripes 1 and 2. V1 and V2, ventral stripes 1 and 2, respectively. Boxes represent melanophore counting areas in D2 and V1.</p

Effects of gender on gene expression levels of some genes associated with the melanogenic pathways in zebrafish as measured by qPCR.

<p><i>Tyrosinase</i> (<i>tyr</i>), <i>tyrosinase-related protein 1a</i> (<i>tyrp1a</i>), <i>tyrosinase-related protein 1b</i> (<i>tyrp1b</i>), <i>dopachrome tautomerase</i> (<i>dct</i>/<i>tyrp2</i>), <i>microphthalmia-associated transcription factor a</i> (<i>mitfa</i>/<i>nacre</i>), <i>sox10</i> (<i>colourless</i>), <i>forkhead transcription factor 3</i> (<i>foxd3</i>), <i>kit receptor tyrosine kinase a</i> (<i>kita</i>, <i>sparse</i>), <i>kitb</i>, <i>melanocortin 1 receptor</i> (<i>mc1r</i>), <i>agouti-signaling protein 1</i> (<i>asip1</i>) and <i>solute carrier family 24 member 5</i> (<i>slc24a5</i>). Asterisks indicate significant differences after t-test (p<0.01) between sexes after Bonferroni's correction.</p

Effects of oral T3 (500 μg/g of food, B) on gene expression levels of genes associated with the melanogenic pathways in zebrafish as measured by qPCR after 7 days (upper panel) and 15 days (lower panel).

<p>After 7 days post-treatment, we found 10 and 9 control females and males, respectively and 10 and 8 T3-treated females and males, respectively. After 15 days post-treatment, we found 6 and 7 control males and females, respectively and 8 and 6 T3-treated males and females, respectively. Asterisks indicate significant T3-induced differences after t-test (p<0.004) after Bonferroni's correction.</p