Atkinson-Leadbeater K et al. (2024), Visual input regulates melanophore differentiat...

XB-ART-60893

Front Cell Dev Biol 2024 Aug 20;12:1437613. doi: 10.3389/fcell.2024.1437613.

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Visual input regulates melanophore differentiation.

Atkinson-Leadbeater K , Bertolesi GE , McFarlane S .

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INTRODUCTION: Developmental processes continue in organisms in which sensory systems have reached functional maturity, however, little research has focused on the influence of sensory input on cell and tissue development. Here, we explored the influence of visual system activity on the development of skin melanophores in Xenopus laevis. METHODS: Melanophore number was measured in X. laevis larvae after the manipulation of visual input through eye removal (enucleation) and/or incubation on a white or black substrate at the time when the visual system becomes functional (stage 40). To determine the developmental process impacted by visual input, migration, proliferation and differentiation of melanophores was assessed. Finally, the role of melatonin in driving melanophore differentiation was explored. RESULTS: Enucleating, or maintaining stage 40 larvae on a black background, results in a pronounced increase in melanophore number in the perioptic region within 24 h. Time lapse analysis revealed that in enucleated larvae new melanophores appear through gradual increase in pigmentation, suggesting unpigmented cells in the perioptic region differentiate into mature melanophores upon reduced visual input. In support, we observed increased expression of melanization genes tyr, tyrp1, and pmel in the perioptic region of enucleated or black background-reared larvae. Conversely, maintaining larvae in full light suppresses melanophore differentiation. Interestingly, an extra-pineal melatonin signal was found to be sufficient and necessary to promote the transition to differentiated melanophores. DISCUSSION: In this study, we found that at the time when the visual system becomes functional, X. laevis larvae possess a population of undifferentiated melanophores that can respond rapidly to changes in the external light environment by undergoing differentiation. Thus, we propose a novel mechanism of environmental influence where external sensory signals influence cell differentiation in a manner that would favor survival.

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Species referenced: Xenopus laevis
Genes referenced: mitf pax3 pmel snai2 sox10 tyr tyrp1 zic1
GO keywords: visually-mediated background adaptation

Phenotypes: Xla Wt + enucleation +GFP + dark (Fig. 1 AB) [+]

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	Figure 1. Enucleation and exposure to a black background dramatically increase perioptic melanophore numbers. (A) The position and size of the counting domains (dashed lines) within each region of analysis is indicated: dorsal midline, perioptic area, flank, and tail (see methods for details). In comparison to controls, melanophore number was unchanged along the dorsal midline, increased dramatically in the perioptic zone, and minimally increased in the flank and tail of enucleated larvae. (dorsal midline: Xcnt = 1.00 0.02; Xenuc = 1.03 0.02; perioptic:; Xcnt = 1.00 0.11; Xenuc = 3.98 0.28; flank: Xcnt = 1.00 0.04; Xenuc = 1.20 0.06; tail: Xcnt = 1.00 0.04, Xenuc = 1.29 0.06; mean s.e.m.; ncnt = 32, nenuc = 33; N = 4) (B) The impact of 24 h exposure to a black background (BB) on perioptic melanophore number was examined. When compared to white background (WB)-exposed controls, BB-treated larvae showed a significant increase in perioptic melanophores, although enucleated larvae displayed an even greater increase in these cells. (Xcnt/WB = 1.00 0.13, XBB = 2.34 0.20, Xenuc = 3.56 0.32; F3,96 = 30.42, p < 0.0001, one-way ANOVA) n. s., not significant, p < 0.05, p < 0.001, **p < 0.0001, unpaired two-tailed Students t-test (A), and Tukeys (B).
	Figure 2. New pigmented cells emerge through de novo pigment production, not migration or proliferation. (A) To track the emergence of new melanophores, enucleated larvae were imaged from 9-19 h post-surgery. A common zone was identified across these images using anatomical landmarks so that individual cells could be tracked over time. Newly emerged pigment cells within the zone were identified (n = 117 melanophores, N = 6). The majority of these (n = 111) appeared de novo as a faint grey dot that darkened over time (white arrows). A few melanophores (n = 6) may have emerged from pigmented cell division (arrowheads). (B) Cell proliferation was inhibited in stage 40 control and enucleated larvae using aphidicolin-hydroxyurea (AH). The numbers of pHH3-positive and pigmented cells were assessed in the perioptic area (white dashed circle) in a blinded fashion after 24 h of AH on a white background. pHH3+ cells in the perioptic region were dramatically reduced in AH-treated larvae (pHH3-positive cells: Xcont = 1.00 0.04, ncont = 24; _Xcont+AH = 0.26 0.03, ncont+AH = 22; Xenuc = 1.12 0.12, nenuc = 24; Xenuc+AH = 0.26 0.04, nenuc+AH = 24; N = 3; F3,90 = 45.77, p < 0.0001, one-way ANOVA). Whereas the enucleation-induced increase in melanophore number was not impacted (pigment-positive cells: Xcont = 1.00 0.08; Xcont+AH = 0.81 0.09; Xenuc = 1.49 0.08; Xenuc+AH = 1.62 0.07; F3,90 = 21.23, p < 0.0001, one-way ANOVA). ***p < 0.0001, Tukeys test.
	Figure 3. Enucleation triggers substantial changes in the expression of tyrp1, tyr and pmel but not mitf in perioptic melanophores. At stage 40, tyrp1, tyr, and pmel are expressed by a small number of perioptic melanophores as assessed by WMISH, whereas mitf expression is rarely visible in individual cells by this method (circular insets show perioptic region; ¯¯¯ X st40 tyrp1 = 13.04 ± 1.36, nst40 tyrp1 = 24, N = 3; ¯¯¯ X st40 tyr = 5.41 ± 1.20, nst40 tyr = 17, N = 2; ¯¯¯ X st40 pmel = 4.81 ± 1.44, nst40 pmel = 16, N = 2; ¯¯¯ X st40 mitf = 0.38 ± 0.18, nst40 mitf = 16; N = 2). After 24 h on a white background with illumination from above (24 h cont./WB), the number of cells expressing tyrp, tyr, and pmel appears reduced, while there continues to be very few mitf expressing cells in the perioptic region ( ¯¯¯ X WB tyrp1 = 2.96 ± 0.61, nst40 tyrp1 = 27; ¯¯¯ X WB tyr = 0.50 ± 0.20, nWB tyr = 18; ¯¯¯ X WB pmel = 2.50 ± 0.67, nWB pmel = 22; ¯¯¯ X WB mitf = 0.89 ± 0.28, nWB mitf = 28). In contrast, WMISH reveals a dramatic increase in tyrp1, tyr, and pmel expressing cells in larvae enucleated at stage 40, prior to being maintained for 24 h in the light on a white background ( ¯¯¯ X Enuc tyrp1 = 36.75 ± 3.12, nEnuc tyrp1 = 24; ¯¯¯ X Enuc tyr = 35.17 ± 3.90, nEnuc tyr = 18; ¯¯¯ X Enuc pmel = 28.82 ± 3.06, nEnuc pmel = 22). However, enucleation does not result in a dramatic increase in mitf-labeled cells in the perioptic region ( ¯¯¯ X Enuc mitf = 3.67 ± 0.78, nEnuc mitf = 27). (One-way ANOVA results: tyrp1:F2,72 = 81.34, p < 0.0001; tyr:F2,50 = 62.23, p < 0.0001; pmel:F2,57 = 50.88, p < 0.0001; mitf:F2,68 = 10.20, p < 0.0001). p < 0.001, *p < 0.0001, n. s., not significant, Tukey’s test.
	Figure 4. Loss of visual input increases tyrp1 expression and perioptic melanophore differentiation. (A–C) tyrp1 mRNA compared to pigmentation for each larva by overlaying WMISH and pigment aggregate images (illustrated in circle and oval schematics for perioptic and flank areas, respectively) at stage 40 (A), after 24 h on a white background (B), and 24 h post-enucleation (C). (D) Average number of pigmented + only, pigmented+/tyrp1+, and tyrp1+ only cells for each condition shown in A-C for the perioptic area and flank. White dashed circle (perioptic region) and oval (flank) indicate analysis regions. (Perioptic-st.40: ¯¯¯ X tyrp1+only = 8.3 ± 1.0, ¯¯¯ X tyrp1+/pig+ = 4.4 ± 0.5, ¯¯¯ X pig+only = 0.4 ± 0.3, nst40 = 26; perioptic-cont: ¯¯¯ X tyrp1+only = 0.4 ± 0.1, ¯¯¯ X tyrp1+/pig+ = 6.3 ± 1.7, ¯¯¯ X pig+only = 6.2 ± 1.2, ncont/WB = 25; perioptic-enuc: ¯¯¯ X tyrp1+only = 0.6 ± 0.2, ¯¯¯ X tyrp1+/pig+ = 32.6 ± 1.5, ¯¯¯ X pig+only = 1.6 ± 0.5, nenuc = 25; N = 3) (Flank-st.40: ¯¯¯ X tyrp1+only = 0.1 ± 0.1, ¯¯¯ X tyrp1+/pig+ = 18.2 ± 1.4, ¯¯¯ X pig+only = 0.1 ± 0.1, nst40 = 16; flank-cont: ¯¯¯ X tyrp1+only = 0.0 ± 0.0, ¯¯¯ X tyrp1+/pig+ = 12.8 ± 0.6, ¯¯¯ X pig+only = 0.0 ± 0.0, ncont/WB = 25; flank-enuc: ¯¯¯ X tyrp1+only = 0.0 ± 0.0, ¯¯¯ X tyrp1+/pig+ = 13.7 ± 0.8, ¯¯¯ X pig+only = 0.1 ± 0.1; nenuc = 25; N = 2) (E) Average pigment aggregate area (perioptic: ¯¯¯ X cnt = 1.00 ± 0.13, ncnt = 15; ¯¯¯ X enuc = 2.49 ± 0.19, nenuc = 19; flank: ¯¯¯ X cnt = 1.00 ± 0.04; ¯¯¯ X enuc = 1.38 ± 0.05; N = 2). ***p < 0.0001, unpaired two-tailed Student’s t-test.
	Figure 5. Undifferentiated melanophores are maintained through larval stages. (A) Stage 40 larvae were exposed to different backgrounds while being illuminated from above for 24–48 h. (B) Larvae were maintain for 24 h on a white background (WB), for 48 h on a white background (WB + WB), for 24 h on a black background (BB), and for 24 h on a white background followed by 24 h on a black background (WB + BB). Perioptic melanophores were quantified (dashed circle in A; ¯¯¯ X WB = 1.00 ± 0.11, nWB = 34; ¯¯¯ X BB = 2.92 ± 0.21, nBB = 35; ¯¯¯ X WB+WB = 1.22 ± 0.13, nWB+WB = 35; ¯¯¯ X WB+BB = 3.04 ± 0.17, nWB+BB = 36; N = 4; F3, 136 = 46.11, p < 0.0001, one-way ANOVA). (C) WMISH for tyrp1 in the perioptic area for the four conditions. ***p < 0.0001, Tukey’s test.
	Figure 6. Optic nerve transection mimics the enucleation-induced increase in melanophore number. (A) Larvae with a unilaterally GFP-labeled optic nerve (on) and optic tectum (ot) underwent sham surgery (cont./WB), single enucleation (sgl. enuc.), double enucleation (dbl. enuc.), or single enucleation with transection of the remaining optic nerve (transection). (B) Images of representative larvae from each surgical condition. The first panel shows presence or absence of GFP-positive optic nerve (arrowheads), second panel shows the presence or absence of GFP-positive retinal ganglion cell axon terminals in the optic tectum (white dashed line), and third panel shows lateral brightfield view of perioptic region for each condition. (C) 24 h after enucleation and/or optic nerve transection or sham surgery, melanophore numbers in the perioptic region (white dashed circle in B, third panel) were compared; ¯¯¯ X cont = 1.00 ± 0.12, ncont = 13; ¯¯¯ X sgl enuc = 1.15 ± 0.14, nsgl enuc = 12; ¯¯¯ X dbl enuc = 2.91 ± 0.32; ndbl enuc = 12; ¯¯¯ X trans = 2.87 ± 0.94, ntrans = 12; N = 2; F3,46 = 23.06, p < 0.0001, one-way ANOVA). ***p < 0.0001, n. s., not significant, Tukey’s test.
	Figure 7. Melatonin signalling is necessary and sufficient to induce perioptic melanophore differentiation. Stage 40 larvae underwent sham surgery and were maintained in the light on a white background or black background, or were enucleated. Larvae from each condition were exposed to control, melatonin, melatonin receptor antagonist (4P-PDOT), or melatonin and 4P-PDOT solutions for a 24-h period. Melanophore numbers in the perioptic region (white dashed circle) were compared; White background: ¯¯¯ X cnt = 1.00 ± 0.4, ncnt = 20; ¯¯¯ X mel = 1.51 ± 0.4, nmel = 20; ¯¯¯ X 4PPDOT = 0.43 ± 0.2, n4PPDOT = 20; ¯¯¯ X mel+4p = 0.50 ± 0.30, nmel+4p = 20; N = 2; F3,79 = 42.34, p < 0.0001, one-way ANOVA; Black background: ¯¯¯ X cnt = 1.54 ± 0.5, ncnt = 20; ¯¯¯ X mel = 1.76 ± 0.6, nmel = 20; ¯¯¯ X 4PPDOT = 0.51 ± 0.3, n4PPDOT = 20; ¯¯¯ X mel+4p = 0.56 ± 0.3, nmel+4p = 20; N = 2; F3,79 = 37.13, p < 0.0001, one-way ANOVA; Enucleation: ¯¯¯ X cnt = 1.70 ± 0.8, ncnt = 20; ¯¯¯ X mel = 1.68 ± 0.5, nmel = 20; ¯¯¯ X 4PPDOT = 0.59 ± 0.3, n4PPDOT = 20; ¯¯¯ X mel+4p = 0.67 ± 0.5, nmel+4p = 20; N = 2; F3,79 = 23.90, p < 0.0001, one-way ANOVA.***p < 0.0001, Tukey’s test.
	Figure 8. Model of vision-mediated melanophore differentiation. At stage 40 (top), as the visual system becomes functional, four related perioptic melanophores exist: 1) differentiated pigment+/tyrp1+, 2) differentiating tyrp1+/pigment-, 3) immature tyrp1-/pigment-awaiting a signal to differentiate, and 4) de-differentiating tyrp1-/pigment+. Light on a white background (bottom left) produces a small increase in pigmented cells, but tyrp1+ cells (pigmented and non-pigmented) decrease. Light on a black background (or 24 h enucleation; bottom right) dramatically increases pigmented and tyrp1+ melanophores. A black background drives differentiation of the tyrp1-/pigment-population into mature pigmented melanophores.