Figure 6. TH promotes xanthophore maturation via scarb1-dependent carotenoid uptake.

(A) Proportions of differentially expressed genes in euthyroid and hypothyroid cells across pseudotime bins. Xanthophores expressed fewer TH-dependent genes than melanophores (expressed gene cutoff = 2% of bin expressing, DEGs are genes with q < 0.05 and fold change >1.5X). Of 160 xanthophore DEGs and 519 melanophore DEGs, only 58 were found to be overlapping. (B) TH-dependent expression of genes related to carotenoid pigmentation in xanthophores. Red bars: q < 0.05, log₂ fold-change ≥2.0. (C) Carotenoid pathway gene expression score was higher in xanthophore lineage cells of euthyroid fish compared to hypothyroid fish (p=1.5E-15, Wilcoxon). By contrast, pteridine pathway gene expression was marginally lower in cells from euthyroid fish (p=0.01). Box-and-whisker plots represent scores across groups (center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers). (D) Carotenoids were detected by HPLC in skin containing xanthophores of euthyroid but not hypothyroid fish (11 SSL). (E)  scarb1 expression in euthyroid and hypothyroid zebrafish (10 SSL). (F) scarb1 mutants lacked mature, yellow xanthophores (12 SSL).

Figure 6—figure supplement 1. Expression of multiple carotenoid-related genes in xanthophores are affected by TH.

UMAP plots of pigment cell clusters colored by expression of TH-dependent genes in xanthophores: gstm.3 (q = 6.9E-99, log₂fold-change = 4.3), plin6 (q = 1.9E-13, log₂fold-change = 1.3), scarb1 (q = 3.5E-11, log₂fold-change = 1.2).

Figure 6—figure supplement 2. TH promotes development of lipid-filled carotenoid droplets in xanthophores.

(A) Carotenoid pigments are normally localized to lipid droplets, the presence of which can be revealed by Oil-red-O staining. Here, a proportion of aox5:palmEGFP+ xanthophores stained ex vivo from euthyroid fish (n = 112 cells) contained lipid (red), whereas xanthophores from hypothyroid fish (n = 48 cells) were never observed to have such lipid contents. (Incidence data in supplementary file Figure 6—figure supplement 1—source data 1.) (B) Ultrastructurally, carotenoids and lipids are detectable as electron-dense carotenoid vesicles (red circles) (Djurdjevič et al., 2015; Granneman et al., 2017; Obika, 1993), which were observed in xanthophores from euthyroid but not hypothyroid fish. N, nucleus. P, pterinosome—the pteridine-containing organelle of xanthophores (Bagnara et al., 1968; Hirata et al., 2003; Matsumoto, 1965; Obika, 1993).

Figure 6—figure supplement 3. scarb1 is specifically involved in xanthophore maturation and is induced by TH.

(A) Scarb1 protein alignment. Zebrafish scarb1 mutants had a 105 bp, in-frame deletion in a conserved region of the protein. (B) scarb1 mutants lacked mature, interstripe xanthophores but had normal stripes and aox5:palmEGFP expression, suggesting that patterning and unpigmented xanthophores were normal. (C) In hypothyroid fish treated with exogenous TH (T4), scarb1 expression was rescued within ~1 d (upper) and carotenoid autofluorescence of xanthophores was recovered within ~2 d (lower).

Figure 6—figure supplement 4. Xanthophores switch yellow pigmentation programs during the larval-to-adult transition.

(A) At 5 dpf, scarb1 mutants had yellow larval xanthophores with wild-type levels of pteridines. (B) Carotenoids were not detectable in EL zebrafish (5 dpf, wild-type; compare to Figure 3D); csf1ra mutants, which lack xanthophores, had HPLC profiles indistinguishable from wild-type. (C) Carotenoid and pteridine pathway signature scores for xanthophores in euthyroid EL and euthyroid and hypothyroid adult scRNA-Seq data sets. Box plots as in Figure 3 with different letters above data indicating significant differences in post hoc comparisons (carotenoid, p<2e-16; pteridine, p=0.01; Tukey HSD). Pteridine signatures between EL, hypothyroid, and euthyroid xanthophores were more similar than carotenoid signatures. (D) Ammonia-induced pteridine fluorescence was present in adult xanthophores of both euthyroid and hypothyroid fish (red arrowheads).