Figure 1.
Model for the molecular action of the melanophore-differentiating and melanogenic genes within melanophores.
Inside melanosomes, the master regulator of melanogenesis, mitf, can regulate the action of tyr, trp2 and trp1, coding for the main enzymes responsible for the synthesis of melanin. Besides, the action of Slc24a5, which is the calcium melanosomal transporter, is crucial for proper melanin synthesis. Pax3 is a key upstream transcription factor in the cascade that can promote or inhibit melanogenesis through transcriptional regulation mitf and cKit, the latter being necessary for melanophore differentiation and responsible for the activation of tyr. Pax3 can also modulate the expression of the two other melanogenic enzymes trp1 and trp2. Mc1r, located to the melanophore membrane, is activated by α-MSH and promotes the activation of tyr. Asip can inhibit the action of mc1r and slc24a5. Hsp70 has been shown to be a negative regulator of casp3 and the latter a negative regulator of mitf. α-MSH, α-melanocyte-stimulating hormone; asip, agouti signaling protein; casp3, caspase 3; cKit, mast/stem cell growth factor receptor Kit; dtc/trp2, L-dopachrome tautomerase; mc1r, melanocyte-stimulating hormone 1 receptor; mitf, microphtalmia-associated transcription factor; pax3, paired box protein Pax-3; sl, somatolactin; slc24a5, sodium/potassium/calcium exchanger; tyr, tyrosinase; trp1, tyrosinase-related protein 1.
Figure 2.
Morphological ontogeny of skin pigmentation in Senegalese sole larvae.
A) 2 dph, B) 5 dph, C) 11 dph, D) 16 dph, E) 19 dph, F) 22 dph, G) 27 dph, H) 33 dph, I) 35 dph, J–K) 41 dph, L–M) 47 dph. Red arrows indicate patches of leucophores and xanthophores. Green arrows show patches of leucophores, xanthophores and melanophores. Note how the allocation of leucophores and xanthophores in dorsal and anal fins at very early stages of larval development serves as referring point for melanophore migration from the dorsal and ventral flanks of the fish to the fins. Red circles show 3 stripes of 5 patches of chromatophores conforming the juvenile pattern of skin color in the ocular side of the fish. Clusters of iridophores delimit these patches of melanophores, xanthophores and leucophores. Fins also follow the same distribution of chromatophores. Green circles delimit the area where iridophores were found in the skin of the blind side of the fish. Scale bar, 600 µm.
Figure 3.
Images of the skin of Senegalese sole revealing the presence, shape, patterning and spatial relationships among melanophores, xanthophores, leucophores and iridophores.
A) 5 dph, B) 22 dph, C–G) 33 dph, H) 35 dph, I-), 40 dph. A) Epidermal melanophores and xanthophores covered the dorsal and ventral flanks of the fish. B) Iridophores were already present in the skin of the ocular side of the fish at the level of the head. C) Detail of the dorsal fin showing melanophores, xanthophores and iridophores. D) Detail of the skin showing the interaction between xanthophores and melanophores. Note how communication between these cells leads to the disintegration of xanthophores. E) Detail of the distribution of chromatophores in the fins. A patch of melanophores, xanthophores and leucophores is surrounded by iridophores. F–J) Detail of the trunk skin of the ocular side showing the distribution pattern of xanthophores and melanophores. Note that the amount of xanthophores relative to melanophores decreased from 33 to 41 dph (F, J). Red arrows, melanophores; blue arrows, xanthophores; yellow arrows, leucophores; green arrows, iridophores; B, E, stereoscopic images; A, C–D, F–J, microscopic images. Scale bars: A, C, G, F, 200 µm; D, H, 250 µm; I, J, 100 µm; E, 500 µm; B, 1 mm.
Figure 4.
Relative amounts of chromatophores (in %) during the ontogeny of the ocular skin pigmentation in Senegalese sole.
A) Relative amount of skin melanophores associated to the processes of melanophore differentiation and melanin synthesis governed by tightly controlled molecular signatures (see also Figs. 6 and 7). Values are expressed as mean ± SD (n = 4). Superscript letters denote significant differences among larvae of different age (One-way ANOVA, P<0.05). Pre-M, premetamorphosis; Pro-M, pro-metamorphosis; Post-M, post-metamorphosis; MD, melanophore differentiation [overall responses of pax3, mitf, mc1r, cKit], MG, melanogenesis [overall responses of tyr, trp1, slc24a5, asip]. B) Relative amount of skin melanophores, xanthophores and iridophores during post-metamorphosis. Values are expressed as mean ± SD (n = 4). Superscript letters denote significant differences between the relative amounts of chromatophores for a given larval age and asterisks indicate significant differences in the amount of a given chromatophore throughout the post-metamorphosis period (One-way ANOVA, P<0.05).
Table 1.
Number of xanthophores (X) associated to one melanophore (M) during the post-metamorphosis period.
Figure 5.
Gene expression patterns of pigmentation related genes during the larval development of Senegalese sole.
Melanocyte-stimulating hormone 1 receptor (mc1r), agouti signaling protein (asip), paired box protein Pax-3 (pax3), mast/stem cell growth factor receptor Kit, (cKit), microphtalmia-associated transcription factor (mitf), tyrosinase (tyr), tyrosinase-related protein 1 (trp1), sodium/potassium/calcium exchanger 5 (slc24a5), caspase 3 (casp3), somatolactin (sl) and heat shock 70 kDa protein (hsp70) Data are represented as means of relative gene expression ± SD (N = 3). Values with a different superscript letter denote significant differences between sampling points (One-way ANOVA, P<0.05).
Figure 6.
Global hierarchical clustering based on similarity of the expression profile for different pigmentation related genes during the larval development of Senegalese sole.
Columns represent the mean data values for each sampling point (days post hatching) and rows represent single genes. Expression level of each gene is represented relative to its median abundance across the different stages and is depicted by a color scale: green, black, and red indicating low, medium, and high relative expression levels, respectively. Colored bars to the right margin indicate the three main gene clusters: blue shows genes highly expressed during pro-metamorphosis stage, orange corresponds to genes highly expressed during pre- and pro-metamorphosis and violet to genes highly expressed at 2 dph and at pro-metamorphosis. The three main stages of the larval development are indicated at the bottom of the figure. Note that the expression of all genes was high during the pro-metamorphosis phase (yellow square), most of genes displaying a shift in their level of expression before and/or after that period. The climax of pigmentation development at the molecular level was observed between 14 and 16 dph (orange square). Changes in gene expression profiles coincided with morphological changes in pigmentation, showing that the climax of metamorphosis was achieved at 16 dph and the end of pro-metamorphosis at 19 dph. The transition from the larval to the adult pattern of skin pigmentation could be clearly observed from 22 dph onwards.
Figure 7.
Gene expression ratios during the larval development of Senegalese sole illustrating the ontogeny of chromatophores.
The asip/mc1r ratio shows the relationship between these genes and reflects the pigmentation patterning of Senegalese sole during development. Its increase during post-metamorphosis coincided with the switch from the bilateral to the flat symmetry and with the appearance of iridophores in the skin of the ocular side. The cKit/pax3 and mitf/pax3 ratios indicate the regulation of cKit and mitf transcription by pax3. Note the initial positive regulation of both genes by pax3 and the subsequent stabilization during the pro-metamorphosis period. The increase of both ratios at post-metamorphosis shows the activation of melanophore migration and melanin synthesis processes during this period. The tyr/mitf and trp1/tyr ratios display opposite profiles during development and correspond well to the melanin production periods. The peaks of tyr/mitf ratio reveal the activation of the melanin synthesis pathway, whereas those of trp1/tyr ratio show the production of melanin. Grey regions indicate the pro-metamorphosis stage.
Table 2.
Target genes used in this study with their accession numbers, main biological processes and amplicon size, primers and hydrolysis probes used in qPCR analyses.