5-Hydroxytryptamine (5-HT) Positively Regulates Pigmentation via Inducing Melanoblast Specification and Melanin Synthesis in Zebrafish Embryos

It has been reported that 5-hydroxytryptamine (5-HT) is related to melanogenesis in mice and melanoma cells. However, the underlying mechanisms of 5-HT in regulating pigmentation remains unknown. In this study, we aim to clarify the regulatory mechanism of 5-HT in the pigmentation of zebrafish embryos and B16F10 cells. Our results show that 5-HT induces the pigmentation of zebrafish embryos in a dosage-dependent manner at concentrations of 0.01–1 mM. Whole mount in situ hybridizations and qRT-PCR in zebrafish embryos indicate that the expression of neural crest cells marker gene sox10 is not changed in embryos treated with 5-HT compared to control group. The expression of mitfa, the marker gene of melanoblasts, is increased in the presence of 5-HT. Furthermore, 5-HT increased the expression of regeneration associated genes, namely kita, mitfa, and dct, after ablation of the melanogenic cells in zebrafish embryos. The experiments in B16F10 cells show that 5-HT promotes melanin synthesis by up-regulating the expression of key proteins MITF, TYR, TRP-1, and TRP-2. Especially, the small molecule inhibitor of PKA signaling, but not AKT and MAPK signaling, attenuates the up-regulation of MITF and TYR resulted from 5-HT induction in B16F10 cells. These results will help us to further understand the regulatory network of vertebrate pigmentation.


Introduction
Melanin, synthesized in vertebrates by a specific type of cell called melanocytes, is the main component of human hair, eyes, and skin color. The melanin distributed in the skin keratinocyte can protect human skin from UV damage [1,2]. Abnormalities of melanin synthesis, metabolism, and melanocytes function in human can lead to related skin diseases, such as albinism, vitiligo caused by insufficient pigment synthesis or loss of pigment, freckles, senile plaques, and even melanoma caused by abnormal accumulation of pigment. There is currently no highly effective treatment for these skin diseases. Therefore, it is of great significance to study the molecular mechanism of regulating pigmentation and to look for drugs regulating or metabolizing pigmentation to understand and treat skin diseases caused by abnormal pigmentation.
Melanocytes are derived from pluripotent neural crest cells and have the same developmental origin as peripheral nervous system [3,4]. The process of early melanocytes differentiation is similar among vertebrates. Neural crest cells specialized by transcription factor SOX10 have the potential

Zebrafish Maintenance and Ethic Statements
All zebrafish lines were purchased from China Zebrafish Resource Center (CZRC, China). All studies involving animal manipulations were approved by the institutional animal use and care committee of Nanjing Ruiying Runze Biopharmaceutical Technology Co., Inc. All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Embryos were incubated at 28.5 • C and staged, according to the description by Kimmel et al. [20].

Drug Treatment
N-Phenylthiourea (PTU), 5-hydroxytryptamine, and 4-(4-morpholinobutylthio) phenol (MoTP) [21] were purchased from Sigma-Aldrich. MoTP was dissolved in dimethyl sulfoxide (DMSO) to make stock solution and then diluted in egg water to 50 µM for all treatments. PTU was dissolved in Milli Q water to make stock solutions and then diluted with egg water to 0.2 mM. Then, 5-HT was dissolved in Milli Q water to make stock solutions and then diluted with egg water to final treatment concentration.

Whole-Mount in situ Hybridization
Whole-mount antisense RNA in situ hybridizations were performed as described [22]. The riboprobes of sox10, kita, mitfa, and dct were previously described. The embryos were fixed in 4% paraformaldehyde (PFA) at 4 • C overnight, then bleached with H 2 O 2 . Digoxigenin-labeled antisense RNA probes were generated in vitro by using the zebrafish sox10, kita, mitfa, and dct cDNA as templates with RNA polymerase.

Measurement of the Pigmenting Activity in the Zebrafish
Synchronized embryos were collected, thirty embryos per well in 6-well plates, and cultured in 5 mL embryo medium. Then, 5-HT and MoTP were dissolved in 0.1% DMSO. In experiments, 0.2 mM PTU was administered from 6 to 35 hpf, then washed and bathed immediately in the medium with indicated concentrations of 5-HT at 35 hpf. The effects on the pigmentation of the zebrafish were observed under the stereomicroscope. All experiments were done at least three times with similar results.

Melanin Content and Tyrosinase Activity Assay
About thirty zebrafish embryos were washed with ice-cold PBS, and sonicated in lysis buffer at 4 • C. Then, lysates were centrifuged at 12,000 rpm for 10 min. Protein concentrations were determined by a BCA kit with bovine serum albumin (BSA) as a standard protocol (Beyotime Technology). Then, 100 µL supernatant containing the same 10 µg total protein was added to each well in a 96-well plate before being mixed with 100 µL 0.1% L-DOPA in 0.1 M PBS (pH 6.8) (M/V). After incubation at 37 • C for 0.5 h, the dopachrome was monitored by measuring the absorbance at 475 nm. Total melanin in the pellet was dissolved in 100 µL of 1 N NaOH with 10% DMSO for 2 h at 80 • C. Then, solubilized melanin was measured at 405 nm. Melanin content was calculated as a percent of the control. All experiments were done at least three times with similar results.

Cell Culture
The B16F10 melanoma cell line was obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco). Subsequently, 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, USA) were placed in a humidified incubator with 5% CO2 and 37 • C.

Statistical Analysis
All data are presented as mean ± SD. The statistical analyses of the results were performed with one-way ANOVA and t test, for comparing all pairs of columns, p < 0.05, 0.01, and 0.001 were accepted as statistically significant.

5-HT Induces Pigmentation in a Dose Dependent Manner in Zebrafish Embryos
To investigate the effect of 5-HT on the pigmentation of zebrafish, a pre-treated PTU model (9-35 hpf) was used in this experiment to reduce pigment background. All the embryos were pretreated with PTU at 9 hpf, then washed and bathed immediately in the fresh egg water at 35 hpf. Different concentration 5-HT (0.01, 0.1, 1 mM) and α-MSH (positive control) ( Figure 1B

5-HT Promotes the Processes of both Melanocytes Development and Melanin Synthesis
Melanocytes of zebrafish embryos, derived from neural crest cells, appear at approximately 24 hpf. Prior to that, the migratory neural crest cells move to the epidermis and differentiate into melanoblast from somitic stage in zebrafish embryos. Along with cell proliferation and differentiation, the melanoblasts express the master regulator Mitf and further induce the other key protein expressions to form functional melanocytes at about 24 hpf. To explore which stage of melanocytes pigmentation was affected by 5-HT, two time windows were examined. Firstly, we investigated the pigmentation effect of 5-HT (9-48 hpf) without treatment of PTU. Compared with control group, 5-HT significantly increases pigmentation of zebrafish embryos at 48 hpf ( Figure 2A-B'). Then the zebrafish embryos were treated with 5-HT from 9 to 24 hpf, when the melanocytes differentiated from neural crest cells. We found that 5-HT increased the number of melanocytes and stimulated the pigmentation in zebrafish embryos at 24 hpf ( Figure 2C,D'). These data indicated that 5-HT may play a positive role in regulating melanocytes development at early stage.
Next, zebrafish embryos were pre-treated with PTU from 9 hpf, then washed intensively and bathed immediately in the fresh egg water at 24 hpf. 5-HT were added and incubated for a further 24 h (until 48 hpf). Similarly, the pigmentation of zebrafish embryos was increased in the presence of 5-HT ( Figure 2E-F'). It seems that 5-HT could promote both melanocytes development and melanin synthesis in zebrafish embryos. Consistent with the result of melanin content ( Figure 1F), the tyrosinase activity ( Figure 1G) was also activated by 5-HT in a dose dependent manner. However, the increase of pigmentation could be resulted from the increase of the numbers of melanocytes or melanin synthesis. Therefore, we next examined the effect on melanocytes development of 5-HT in zebrafish embryos. Because of the most obvious pigmentation of 1 mM 5-HT, we chose this concentration for further study.

5-HT Promotes the Processes of both Melanocytes Development and Melanin Synthesis
Melanocytes of zebrafish embryos, derived from neural crest cells, appear at approximately 24 hpf. Prior to that, the migratory neural crest cells move to the epidermis and differentiate into melanoblast from somitic stage in zebrafish embryos. Along with cell proliferation and differentiation, the melanoblasts express the master regulator Mitf and further induce the other key protein expressions to form functional melanocytes at about 24 hpf. To explore which stage of melanocytes pigmentation was affected by 5-HT, two time windows were examined. Firstly, we investigated the pigmentation effect of 5-HT (9-48 hpf) without treatment of PTU. Compared with control group, 5-HT significantly increases pigmentation of zebrafish embryos at 48 hpf ( Figure 2A-B'). Then the zebrafish embryos were treated with 5-HT from 9 to 24 hpf, when the melanocytes differentiated from neural crest cells. We found that 5-HT increased the number of melanocytes and stimulated the pigmentation in zebrafish embryos at 24 hpf ( Figure 2C,D'). These data indicated that 5-HT may play a positive role in regulating melanocytes development at early stage.

5-HT Facilitate the Melanoblast Specification from Neural Crest Cells
To clarify how 5-HT affect the melanocytes development in zebrafish embryos, whole mount in situ hybridization and quantative real time PCR were used to test the expression level of marker genes of neural crest cells (sox10), melanoblast (mitfa), and differentiated melanocytes (dct) [23]. To investigate whether the neural crest cells were affected by 5-HT, the expression of sox10 was examined in presence of 5-HT from 9 to 18 hpf. The results showed that the expression of sox10 was not changed in the zebrafish embryos treated with 5-HT ( Figure 3A,B,I). This result indicated that 5-HT was not sufficient for the cell identity of neural crest cells. However, the expression of melanoblast marker gene, mitfa was increased in eyes and dorsal at 24 hpf with 5-HT treatment from 18 to 24 hpf ( Figure 3C,D,I). That means 5-HT may play a key role in regulating neural crest differentiate into melanoblasts.
Interestingly, the expression of differentiated melanocytes marker gene, dct was not changed at 48 hpf with 5-HT treatment from 24 to 48 hpf ( Figure 3E,F,I). However, the expression of dct was increased at 48 hpf when zebrafish embryos treated with 5-HT from 9 to 48 hpf ( Figure 3G-I).
Meanwhile, the number of dct+ cells was not changed in the embryos treated with 5-HT or PTU ( Figure 3G, Supplementary Figure S1). In summary, these results suggest that 5-HT plays a key role specifically in regulating melanoblast specification from neural crest cells but does not affect the cell identity of neural crest cells and the melanocytes differentiated from melanoblasts. Next, zebrafish embryos were pre-treated with PTU from 9 hpf, then washed intensively and bathed immediately in the fresh egg water at 24 hpf. 5-HT were added and incubated for a further 24 h (until 48 hpf). Similarly, the pigmentation of zebrafish embryos was increased in the presence of 5-HT ( Figure 2E-F'). It seems that 5-HT could promote both melanocytes development and melanin synthesis in zebrafish embryos.

5-HT Facilitate the Melanoblast Specification from Neural Crest Cells
To clarify how 5-HT affect the melanocytes development in zebrafish embryos, whole mount in situ hybridization and quantative real time PCR were used to test the expression level of marker genes of neural crest cells (sox10), melanoblast (mitfa), and differentiated melanocytes (dct) [23]. To investigate whether the neural crest cells were affected by 5-HT, the expression of sox10 was examined in presence of 5-HT from 9 to 18 hpf. The results showed that the expression of sox10 was not changed in the zebrafish embryos treated with 5-HT ( Figure 3A,B,I). This result indicated that 5-HT was not sufficient for the cell identity of neural crest cells. However, the expression of melanoblast marker gene, mitfa was increased in eyes and dorsal at 24 hpf with 5-HT treatment from 18 to 24 hpf ( Figure 3C,D,I). That means 5-HT may play a key role in regulating neural crest differentiate into melanoblasts.

5-HT Prompts Regeneration of Larval Zebrafish Melanocytes
Regeneration is one of the most popular concern of modern medicine. The regeneration of melanocytes brings hope to many skin diseases with melanocytes dysfunction or apoptosis, such as vitiligo. How to make the melanocytes cease to quiescent condition and reenter the cell cycle is the biggest challenge in melanocytes regeneration. It has been reported that the Kit receptor tyrosine kinase is essential for the regeneration of zebrafish larval melanocytes [24].
Since the 5-HT showed a promotion effect on melanogenic lineage, we speculated that whether 5-HT could induce the regeneration of melanocytes. In this study, the small molecule MoTP was used to ablate the melanocytes and melanoblast. The melanocytotoxicity was reported mediated via tyrosinase activity, presumably to convert MoTP to cytotoxic quinone species. The zebrafish embryos were pretreated with 50 µM MoTP from 9 to 35 hpf to eliminate most of the melanocytes at early stage. Compared to blank group, MoTP treatment resulted in decrease of melanoblast markers (kita+ and mitfa+) and differentiated melanocytes (dct+) at 60 hpf in zebrafish embryos ( Figure  4B   Interestingly, the expression of differentiated melanocytes marker gene, dct was not changed at 48 hpf with 5-HT treatment from 24 to 48 hpf ( Figure 3E,F,I). However, the expression of dct was increased at 48 hpf when zebrafish embryos treated with 5-HT from 9 to 48 hpf ( Figure 3G-I). Meanwhile, the number of dct+ cells was not changed in the embryos treated with 5-HT or PTU ( Figure 3G, Supplementary Figure S1). In summary, these results suggest that 5-HT plays a key role specifically in regulating melanoblast specification from neural crest cells but does not affect the cell identity of neural crest cells and the melanocytes differentiated from melanoblasts.

5-HT Prompts Regeneration of Larval Zebrafish Melanocytes
Regeneration is one of the most popular concern of modern medicine. The regeneration of melanocytes brings hope to many skin diseases with melanocytes dysfunction or apoptosis, such as vitiligo. How to make the melanocytes cease to quiescent condition and reenter the cell cycle is the biggest challenge in melanocytes regeneration. It has been reported that the Kit receptor tyrosine kinase is essential for the regeneration of zebrafish larval melanocytes [24].
Since the 5-HT showed a promotion effect on melanogenic lineage, we speculated that whether 5-HT could induce the regeneration of melanocytes. In this study, the small molecule MoTP was used to ablate the melanocytes and melanoblast. The melanocytotoxicity was reported mediated via tyrosinase activity, presumably to convert MoTP to cytotoxic quinone species. The zebrafish embryos were pretreated with 50 µM MoTP from 9 to 35 hpf to eliminate most of the melanocytes at early stage. Compared to blank group, MoTP treatment resulted in decrease of melanoblast markers (kita+ and mitfa+) and differentiated melanocytes (dct+) at 60 hpf in zebrafish embryos ( Figure 4B,B',G,G',L,L'). When different concentrations of 5-HT were added into the medium of zebrafish embryos until 60 hpf. Surprisingly, we observed the increased expression of kita ( Figure 4A-E

5-HT Increases the Expression of MITF and TYR by Activating the PKA/p-CREB Signaling in Melanoma Cells
Usually, 5-HT plays a role in different cells type through binding to the specific 5-HT receptor(s) on the cell membrane. Moreover, 5-HT receptors are a big G protein coupled receptor family, which include seven subfamilies from HTR1-7. The signaling transduction mediated by G protein involve cAMP/PKA, MAPK, DAG/PKC, and so on. At the start of this study, we found that 5-HT regulates not only melanocytes development, but also melanin synthesis. At the same time, the expression of differentiated melanocytes marker gene dct increased in 5-HT treated zebrafish embryos from 9 to 48 hpf without increase of melanocytes number, we surmised that 5-HT could regulate the melanin synthesis in differentiated melanocytes. Therefore, next, we want to clarify the key signaling pathway regulating melanin synthesis in the presence of 5-HT.
We examined the effect of 5-HT on the expression of key proteins related to melanogenesis, MITF, TYR, TRP1 and TRP-2 in melanocytes. The results showed that the expression levels of these four proteins were enhanced significantly at 48 h after 5-HT treated in B16F10 melanoma cells ( Figure  5A, Supplementary Figure S2A-D). Furthermore, key proteins of PKA, AKT, and MAPK signaling pathways, p-CREB, p-AKT, p-p38, p-JNK, and p-ERK were tested by Western blotting. The results showed that p-CREB protein level was increased in the B16F10 cells with 5-HT treatment in a dose dependent manner (Figure 5B, S2E). The inhibitor of PKA signaling, H89 showed an inhibiting effect on the expression increase of MITF and TYR with 5-HT treatment ( Figure 5C, Supplementary Figure  S2F-G).
Similarly, p-AKT and p-ERK protein levels were increased by 5-HT treatment in B16F10

5-HT Increases the Expression of MITF and TYR by Activating the PKA/p-CREB Signaling in Melanoma Cells
Usually, 5-HT plays a role in different cells type through binding to the specific 5-HT receptor(s) on the cell membrane. Moreover, 5-HT receptors are a big G protein coupled receptor family, which include seven subfamilies from HTR1-7. The signaling transduction mediated by G protein involve cAMP/PKA, MAPK, DAG/PKC, and so on. At the start of this study, we found that 5-HT regulates not only melanocytes development, but also melanin synthesis. At the same time, the expression of differentiated melanocytes marker gene dct increased in 5-HT treated zebrafish embryos from 9 to 48 hpf without increase of melanocytes number, we surmised that 5-HT could regulate the melanin synthesis in differentiated melanocytes. Therefore, next, we want to clarify the key signaling pathway regulating melanin synthesis in the presence of 5-HT.
We examined the effect of 5-HT on the expression of key proteins related to melanogenesis, MITF, TYR, TRP1 and TRP-2 in melanocytes. The results showed that the expression levels of these four proteins were enhanced significantly at 48 h after 5-HT treated in B16F10 melanoma cells ( Figure 5A, Supplementary Figure S2A-D). Furthermore, key proteins of PKA, AKT, and MAPK signaling pathways, p-CREB, p-AKT, p-p38, p-JNK, and p-ERK were tested by Western blotting. The results showed that p-CREB protein level was increased in the B16F10 cells with 5-HT treatment in a dose dependent manner ( Figure 5B, Figure S2E). The inhibitor of PKA signaling, H89 showed an inhibiting effect on the expression increase of MITF and TYR with 5-HT treatment ( Figure

Discussion
Despite the link between psychological stress and skin problems has long been well known, its specific association has never been fully clarified [25]. The function of those neurotransmitters in the physiological and pathological state of the skin remains poorly understood. Moreover, 5-HT is a phylogenetically ancient signaling molecule and neurotransmitter, which actions are mediated through 5-HT receptors [26,27]. It has been reported that 5-HT receptors are widely detected on mammalian dermal fibroblasts and melanocytes. Mammalian skin cells have the capability to produce and metabolize 5-HT. Further, 5-HT and its metabolite melatonin were associated with the regulation of numerous skin physiological functions [28,29].
In our previous study, the level of 5-HT in vitiligo patients' serum was significantly decreased. Mice with chronic unpredictable mild stress exhibit hypopigmentation and a decrease of 5-HT level in both serum and skin. Fluoxetine, the classical 5-HT reuptake inhibitor, shows the ability to promote melanogenesis in melanoma cells and mice with psychological stress. All those studies indicate the crucial role of 5-HT in regulating the skin melanocytes maintenance and function. However, the underlying mechanisms have been rarely reported.
Mechanisms for specifying melanoblast from neural crest cells that involve upregulation of the microphthalmia transcription factor, Mitf (mitfa in zebrafish) in neural crest cells are conserved in mice, chick and zebrafish [30,31]. Transcription factor Sox10 is required for the expression of mitf in neural crest cells. When melanoblasts are specified, Mitf transcriptionally activates additional melanocytes differentiation and migration genes, including the melanin synthesis enzymes, dct, tyr, Similarly, p-AKT and p-ERK protein levels were increased by 5-HT treatment in B16F10 melanoma cells ( Figure 5B, Supplementary Figures S3A-S4A). This is consistent with the reality that 5-HT is a non-selective agonist of 5-HT receptors. However, the inhibitors of PI3K/AKT and ERK, LY294002 and PD98059, could not affect the increased expression of MITF and TYR by 5-HT treatment in B16F10 melanoma cells (Supplementary Figures S3 and S4).
In short, 5-HT may play a key role in promoting pigmentation through up-regulating the expression of melanogenesis related key proteins, MITF and TYR, by activating PKA/p-CREB specifically. However, which 5-HT receptor mediate this effect require further study.

Discussion
Despite the link between psychological stress and skin problems has long been well known, its specific association has never been fully clarified [25]. The function of those neurotransmitters in the physiological and pathological state of the skin remains poorly understood. Moreover, 5-HT is a phylogenetically ancient signaling molecule and neurotransmitter, which actions are mediated through 5-HT receptors [26,27]. It has been reported that 5-HT receptors are widely detected on mammalian dermal fibroblasts and melanocytes. Mammalian skin cells have the capability to produce and metabolize 5-HT. Further, 5-HT and its metabolite melatonin were associated with the regulation of numerous skin physiological functions [28,29].
In our previous study, the level of 5-HT in vitiligo patients' serum was significantly decreased. Mice with chronic unpredictable mild stress exhibit hypopigmentation and a decrease of 5-HT level in both serum and skin. Fluoxetine, the classical 5-HT reuptake inhibitor, shows the ability to promote melanogenesis in melanoma cells and mice with psychological stress. All those studies indicate the crucial role of 5-HT in regulating the skin melanocytes maintenance and function. However, the underlying mechanisms have been rarely reported.
Mechanisms for specifying melanoblast from neural crest cells that involve upregulation of the microphthalmia transcription factor, Mitf (mitfa in zebrafish) in neural crest cells are conserved in mice, chick and zebrafish [30,31]. Transcription factor Sox10 is required for the expression of mitf in neural crest cells. When melanoblasts are specified, Mitf transcriptionally activates additional melanocytes differentiation and migration genes, including the melanin synthesis enzymes, dct, tyr, and trp1, and the melanocytes migration and survival gene, kit. Other signaling such as WNT and BMP are also involved in the melanocytes development [31,32]. Therefore, the effect on the cell fate determination of melanogenic lineage induced by 5-HT during embryonic stage and regeneration condition is what we want to illustrate in this research.
Firstly, 5-HT promoted pigmentation in a dose dependent manner in zebrafish embryos. Through further investigation, we found 5-HT had no effect on neural crest cells marked by transcription factor sox10, at 18 hpf in zebrafish embryos. However, the expression of melanoblast marker, mitfa, was increased at 24 hpf. When we treated the zebrafish embryos with 5-HT from 24 to 48 hpf, the expression of differentiated melanocytes marker gene dct was not affected. These results suggest 5-HT specifically play a key role in regulating melanoblast specification from neural crest cells. Secondly, we used small molecule MoTP to ablate the early melanoblast cells and differentiated melanocytes. After the ablation of melanogenic cells, the expression of essential genes for melanocytes regeneration, such as kita and mitfa, were increased with 5-HT treatment in zebrafish. The therapeutic method that pushes the resident progenitor cells to differentiate into melanogenic cells will greatly contribute to the treatment of skin diseases like vitiligo. However, the detailed role of 5-HT in regulating follicle stem cell cycle in mammals still needs to be elucidated.
Finally, we identified the downstream signaling activated by 5-HT in regulating melanin synthesis in differentiated melanocytes. B16F10 melanoma cell line is widely used for regulatory studies as an in vitro model. Here, we treated the B16F10 cells with 5-HT. The expression of the melanogenesis related key proteins, MITF and TYR were increased in the melanocytes with 5-HT treatment. Lots of signaling pathways, such as MAPK, PKA, AKT, and so on, have been reported to participate in the process of melanogenesis [33][34][35]. The MAPK signaling pathway, including p38, JNK, and ERK, is an important pathway involved in melanogenesis. Activations of the ERK and JNK pathways were related to the downregulation of melanogenesis [36,37]. P38 is involved in melanogenesis induced by UV irradiation. In this study, our data showed that 5-HT regulated the expression of MITF and TYR in B16F10 melanoma cells by specifically activating PKA/p-CREB signaling pathways. In terms of previous studies, HTR1A/2A are included in the regulation of melanogenesis. However, the crucial receptor of 5-HT in regulating melanocytes development and melanin synthesis needs to be identified.

Conclusions
In conclusion, our study showed that 5-HT induced the melanoblast specification from neural crest cells and regeneration in zebrafish embryos. In addition, 5-HT may prompt melanin synthesis by up-regulating the expression of melanogenic master regulator MITF and rate-limiting enzyme TYR. The protein kinase A signaling was further proven to be the key downstream signal of 5-HT in regulating melanogenesis in a melanoma cell line. These results will help to comprehensively understand the regulatory network of 5-HT in melanocytes development, regeneration, and function.
Author Contributions: L.L., M.Z., J.D., and M.C. performed the research. Y.Y. and L.L. wrote the paper. Y.Y., J.S., and L.L. designed the research study. Y.Y. contributed to the development of methodology. L.L. analyzed the western blot and mRNA data. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the National Natural Science Foundation of China (No. 81874331).

Conflicts of Interest:
The authors declare no conflict of interest.