Background: Vertebrates use different pigmentation strategies to adapt to various environments. A large amount of research has been done on disclosing the mechanisms of pigmentation strategies in vertebrates either under light, or, living in constant darkness. However, less attention has been paid to non-obligate, darkness dwellers. Red-spotted toothed toads Oreolalax rhodostigmatus (Megophryidae; Anura) from the karst mountainous region of southwestern China are non-obligate cave dwellers. Most tadpoles of the species possess transparent skin as they inhabit the dark karst caves. But remarkably, the transparent tadpoles can darken just within 15 h once exposed to light. Obviously, it is very significant to reveal molecular mechanisms of the unexpected rapid-darkening phenomenon. Results: We compared the transcriptomes of O. rhodostigmatus tadpoles with different durations of light exposure to investigate the cellular processes and potential regulation signals for their light-induced rapid darkening. Genes involved in melanogenesis (i.e. TYR, TYRP1 and DCT) and melanocyte proliferation, as well as their transcriptional factor (MITF), showed light-induced transcription, suggesting a dominating role of morphological color change (MCC) in this process. Transcription of genes related to growth factor, MAPK and PI3K-Akt pathways increased with time of light exposure, suggesting that light could induce significant growth signal, which might facilitate the rapid skin darkening. Most importantly, an in-frame deletion of four residues was identified in O. rhodostigmatus melanocortin-1 receptor (MC1R), a critical receptor in MCC. This deletion results in a more negatively charged ligand pocket with three stereo-tandem aspartate residues. Such structural changes likely decrease the constitutive activity of MC1R, but increase its ligands-dependent activity, thus coordinating pigment regression and rapid melanogenesis in the dark and light, respectively. Conclusion: Our study suggested that rapid MCC was responsible for the light-induced rapid darkening of O. rhodostigmatus tadpoles. Genetic mutations of MC1R in them could explain how these non-obligate cave dwellers coordinate pigment regression and robust melanogenesis in darkness and light, respectively. To our knowledge, this is the first study that reports the association between pigmentation phenotype adaptation and MC1R mutations in amphibians and/or in non-obligate cave dwellers. Keywords: Cave dweller, Melanocortin 1 receptor, Pigment regression, Melanogenesis, Morphological color change * Correspondence: firstname.lastname@example.org; email@example.com CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhu et al. BMC Genomics (2018) 19:422 Page 2 of 13 Background precursor proopiomelanocortin protein (POMC), α-MSH Vertebrates use different pigmentation strategies to binds to its receptor, melanocortin-1 receptor (MC1R), adapt to various environments [1, 2]. For species living expressed on the cell membrane of chromatophores to in environments with diurnal and seasonal changing stimulate pigment dispersion . All the three major chro- light, skin pigment is necessary for preventing optical matophore types (melanophore, red erythrophore and yel- damage, and pigmentation configurations matching to low xanthophore) share similar PCC regulation patterns the background are beneficial for their survival [3–6]. and mechanisms in frogs . MCC is characterized by pig- On the contrary, for species living in constant darkness ment synthesis, chromatophore proliferation and chro- (i.e.insidethe caves),pigment regression is acom- matophore. This kind of color changes is relatively slow, mon survival strategy [7–12], as pigmentation can be generally occurring over days or weeks [2, 6, 21]. The mo- resource-consuming . In these animals, the cap- lecular processes of MCC have been well studied in acity to generate pigmentation is completely or partly melanocytes in mammals. Melanin synthesis in melano- lost [8–12]. Beside these two well-studied categories, cytes, called melanogenesis, also requires the binding of there is the third category of animals, who may experience α-MSH to MC1R, which activate the transcription of both dark and light conditions in their life histories (i.e. microphthalmia-associated transcription factor (MITF), the non-obligate cave dwellers). However, their pigmentation key transcriptional factor in melanocyte differentiation and strategy and mechanisms are less concerned. melanogenesis [22, 23]. Meanwhile, activation of MAPK Red-spotted toothed toad species Oreolalax rhodostig- and PI3K-Akt pathways, which activated by growth factors matus (Megophryidae, Anura) is a typical non-obligate released from keratinocyte in response to ultraviolet radi- cave dweller, which is distributed in the karst mountain- ation, is responsible for phosphorylating MITF protein to ous region (altitude 500–2400 m. a. s. l.) of southwestern its activation form [24–26]. Then, MITF activate melano- China . Most tadpole populations of the species gen- cyte differentiation and expression of tyrosinase (TYR), erally live in dark caves for several years with transpar- tyrosinase-related protein 1 (TYRP1) and dopachrome tau- ent skin as pigment regression, but meanwhile, some tomerase (DCT) for melanin synthesis. The molecular pro- tadpole populations could also inhabit the “out-side and cesses of MCC in frogs are likely similar to that of light” streams and possess black brown body color simi- mammals, as α-MSH, receptor tyrosine kinase and MITF lar to their juvenile and adult frogs usually foraging out- has been reported for early development of chromato- side the caves and generally possessing dark black body phores in fishes and frogs [26–33]. color [15, 16]. The most noticeable thing is that the In gene levels, the functional mutations in genes re- transparent O. rhodostigmatus tadpoles could undergo sponsible for regulation of pigmentation are widely iden- fast and drastic darkening once exposed to the sunlight, tified in vertebrates. For example, defects in genes with vast array of black spots presented within 4 h and involved in melanin biosynthesis (i.e., OCA2 and TYR) whole-body brown to black skin color within 15 h were always identified in cave fishes [8, 11, 12, 34]. Des- (Fig. 1a-f). The remarkable pigmentation capacity of the pite the large number of potential targets, only a handful species indicates that their body probably has an “optical of genes have been identified to contribute to genetic switch” for pigmentation to coordinate the opposite pig- color adaptation in many animal taxa. Of these, MC1R mentation requirements in darkness and light. Neverthe- are among the most widely studied pigmentation genes less, to present, there is no work focusing on disclosing in mammals, birds, reptiles and fish , and some stud- the mechanisms of this fascinating phenomena. ies now show a link between variation in MC1R and pig- Generally, light can induce physiological color change mentation in numerous vertebrates [35–39]. MC1R is a (PCC) and morphological color change (MCC) in ani- member of the G protein-coupled receptor superfamily, mals. PCC is defined as rapid color change (within consisted of 7 transmembrane fragments, 3 extracellular several hours) facilitated by the dispersion of pigment loops, 3 intracellular loops, 1 N-terminus and 1 C-terminus. granules (color deepening) and aggregation (color fad- Known mutations are largely interspersed throughout ing) within chromatophores without pigment synthesis the transmembrane fragments and loops. The mem- [17–19]. It is commonly observed in the color change of brane/extracellular junctions of the second and third fish, amphibians and reptiles. PCC is mainly regulated transmembrane domains (M/EJTD), which are nega- by hormones, whose release is responsive to light . tively charged, is likely the site of electrostatic inter- Hormones inducing pigment dispersion in frogs includes action with the arginine residue in α-MSH . alpha-melanocyte-stimulating hormone (α-MSH), adreno- Mutations introducing basic residues or eliminating corticotropic hormone (ACTH) and melanin-concentrating acid residues in this region always result in enhanced hormone(MCH),while thoseinducepigment aggregation constitutive activity of MC1R in manner of ligand include melatonin and adrenaline . α-MSH is the most mimic , but also reduced agonist binding activity studied hormone involved in PCC. After incised from its . Interestingly, though melanistic phenotypes in a Zhu et al. BMC Genomics (2018) 19:422 Page 3 of 13 Fig. 1 Phenotypes of O. rhodostigmatus tadpoles and transcriptomes analyses flow. a-c, Phenotypes of O. rhodostigmatus tadpoles in control group, short-term exposed group and long-term exposed group. d-f, Dorsal skin of O. rhodostigmatus tadpoles, blue arrows indicate typical black spots in short-term exposed larvae. g, Analyses flow of transcriptome data. h, Correlations of genes expression pattern between samples. A higher squared correlation coefficient (R ,0–1) indicates more similar expression profiles between samples certain species are always associated with basic resi- were analyzed by sequence alignments and protein hom- dues introducing and/or acid residues eliminating in ologous modeling to screen functional evolution respon- these regions [41–50], pigment regression or pale siblefor thepigmentationstrategyof O. rhodostigmatus. phenotypes have not been identified to be granted by mutations acidifying this region [9, 51, 52]. As note, Results there is no report in amphibians in regard to the as- Summary of transcriptome assembly and transcript sociations between color adaptation and variations of annotation MC1R genes. A total of 249,088 unigenes were obtained from 13 Hence, in this study, we try to uncover the mechanisms cDNA libraries, with their sequencing quality summa- of the remarkable rapid darkening in O. rhodostigmatus rized in Additional file 1: Table S1. The mean length and tadpoles using comparative transcriptomics. Firstly, the col- N50 of unigenes were 1148 bp and 1928 bp, respectively oration type was determined by analyzing the changes of (see Additional file 2: Figure S1 for length distribution). expression pattern of genes involved in pigmentation with In total, 76,223, 57,186, 51,598, 81,733, 77,971, 81,074 light exposure duration. Secondly, major light-induced and 36,301 unigenes were annotated in NR, NT, KO, transcriptional events were highlighted with gene differen- SwissProt, PFAM, GO and KOG data bases, respectively tial expression and functional enrichment analyses, and (see Additional file 3: Table S2 for annotation details). their potential contributions to light-induced rapid darken- Their expression levels (presented as FPKM) were sum- ing in O. rhodostigmatus were discussed. Thirdly, genes po- marized in Additional file 4: Table S3. Overall, the tentially contributing to genetic adaptation of coloration intra-group correlations are higher than inter-group Zhu et al. BMC Genomics (2018) 19:422 Page 4 of 13 ones in this study (Fig. 1g), supporting the validity of genes. Most growth factor-related genes identified in our our transcriptome data. study are transcriptionally responsive to light exposure A total of 1662 light inducible genes were identified (Fig. 4c & d). Those related to epidermal growth factor (fdr < 0.05, one-way ANOVA). Among these, 213 ones (EGF), platelet-derived growth factor (PDGF), fibroblast show upregulation in subsequent pairwise comparisons: growth factor (FGF), hepatocyte growth factor (HGF), “short-term exposed vs control” and “long-term exposed and mast/stem cell growth factor (M/SCGF) showed vs short-term exposed” (Fig. 1h). These two groups of light-depended transcription, while those related to genes were respectively queried against KEGG database insulin-like growth factor mainly reached their highest for enrichment analysis, and they shared most signifi- expressions after short-term light exposure (Fig. 4c & d). cantly enriched pathways (see Additional file 5: Table S4 and Additional file 6: Table S5 for details), suggesting Structural and functional change of O. rhodostigmatus that the results of enrichment analyses were not sensi- MC1R tive to the thresholds of differentially expressed genes. The sequences of O. rhodostigmatus α-MSH, MC1R, MC4R, MC5R, MCHR, agouti, TYR, TYRP1 and DCT Coloration type of light-induced darkening were aligned with that of fishes, other amphibians, rep- Transcription of effector genes in melanogenesis (i.e., tiles, birds and mammals. Prominent difference was TYR, TYRP1 and DCT) and marker genes of melanocyte identified only in O. rhodostigmatus MC1R. The length (PMEL isoform X1, melanoma antigen recognized by of ECL1 of MC1R was conserved across vertebrates, T-cells 1 isoform X1/MELANA, Melanoregulin X1, however, a deletion of four amino acids in this region Melanoregulin X3, and premelanosome protein pre- was detected in its O. rhodostigmatus MC1R (Fig. 5). cursor/PMEL) were increased with light exposure in Such a deletion changed the structure of the M/EJTD O. rhodostigmatus tadpoles (Fig. 2a). Their transcrip- and resulted in a longer third transmembrane helix in O. tional factor, MITF, was also transcriptionally activated by rhodostigmatus MC1R than that of other species, as sug- light exposure, with most of its transcripts peaked after gested by the 3D models (Additional file 7: Figure S2). short-term light exposure (Fig. 3). Xanthophores and ery- Since the charged amino acids responsible for agonist throphores show no sign of response to light in manner of binding and constitutive activation of MC1R, we further MCC due to genes involved in synthesis or metabolism of analyzed the charge property of this region. Interestingly, carotenoids (beta, beta-carotene 9′,10′-oxygenase isoform fragment deletion in O. rhodostigmatus MC1R covered X1/BCO2 and beta, beta-carotene 15,15′-monooxygen- the sites with high frequency of positively charged amino ase/BCMO1) and pteridines (dihydropteridine reductase/ acids (Fig. 6a), and the net charge of this region is −5in DHPR, GTP cyclohydrolase 1 feedback regulatory pro- O. rhodostigmatus, more negatively charged than other tein/GCHFR, GTP cyclohydrolase 1/GCH1, sepiapterin frogs (Fig. 6b). On the other hand, fragment deletion in reductase/SPR and xanthine dehydrogenase/oxidase/ O. rhodostigmatus MC1R resulted in a “Asp× × ×Asp× × XDH/XOD) were not upregulated, or even downregulated ×Asp× × ×” sequence mode, and these three negatively (Fig. 2b). In addition, neither MC1R, MC4R, MC5R, charged aspartate residues were aligned towards the lig- MCH receptor (MCHR), PMOC and agouti signal peptide and pocket of MC1R (Fig. 6c), quite different with the (ASIP, antagonist of MC1R), which participate in signal distribution of aspartate residues in other MC1Rs transduction of pigment dispersion/aggregation, nor mela- (Additional file 7: Figure S2). In combination with the nophilin proteins (excepting melanophilin 5), which were elongated third helix of MC1R, these three stereo-tandem responsible for transport of melanosome, were transcrip- aspartate residues likely performed as a trap for positively tionally upregulated by light (Fig. 2c). charged ligands. Interestingly, both MSH and ACTH, who are positively charged at pH 7.0 (Fig. 6d-e) with isoelectric Light-induced major transcriptional events points (IEP) were higher than 9.0. Here, we focused on the results of enrichment analysis based on the 213 light-inducible genes. The top 20 Discussion (sorted by q value) enriched pathways/processes mainly Molecular processes involved in light-induced rapid skin referred to cancer and signal transduction (Fig. 4a). darkening Genes enriched in these pathways/processes were fur- Though the genes involved in synthesis of melanin, ca- ther compared and integrated into a consensus module rotenoids and pteridines are all transcribed in O. rhodos- manually. This module was composed of growth factor tigmatus tadpoles, only melanin-related genes showed receptors (GFRs), core components of MAPK signal increased transcription with light exposure (Fig. 2). It pathway, and core components of PI3K-Akt signal path- suggested that only melanocytes were involved in skin way (Fig. 4b). In addition, growth factor-related genes darkening in O. rhodostigmatus tadpoles, as melanin has constituted the largest gene group among light inducible a superiority on UV-light absorption in comparing with Zhu et al. BMC Genomics (2018) 19:422 Page 5 of 13 Fig. 2 Transcriptional variation of coloration related genes in response to light exposure. a, Melanogenesis effector genes and melanocyte marker genes. b, Genes involved in synthesis of carotenoids and pteridines. c, Genes involved in PCC. Each column represents a mean ± SE, ***: p < 0.001, **: p < 0.01, *: p < 0.05 (T-test). TYRP1, tyrosinase-related protein 1; TYR, tyrosinase; DCT, dopachrome tautomerase; MLANA, melanoma antigen recognized by T-cells; Melanoregulin X1, melanoregulin isoform X1; Melanoregulin X3, melanoregulin isoform X3; PMEL, premelanosome protein precursor; PMEL X1, melanocyte protein PMEL isoform X1; DHPR, dihydropteridine reductase; GCHFR, GTP cyclohydrolase 1 feedback regulatory protein; GCH1, GTP cyclohydrolase 1; SPR, sepiapterin reductase; XDH/XOD, xanthine dehydrogenase/oxidase; BCO2, beta,beta-carotene 9′,10′-oxygenase; BCMO1, beta,beta-carotene 15,15′-monooxygenase. MC1R, melanocyte-stimulating hormone receptor / melanocortin receptor 1; MCHR, melanin-concentrating hormone receptor; Agouti, agouti signaling peptide; MC4R, melanocortin receptor 4; MC5R, melanocortin receptor 5; POMC, pro-opiomelanocortin carotenoids and pteridines. Both PCC and MCC may be amphibians , and in most studied cases, activation of involved in melanin-based pigmentation. Generally MCC is always accompanied by PCC, as factors inducing speaking, PCC is more active and rapid than MCC in MCC can also activate PCC . However, in O. rhodos- response to environmental brightness in fish and tigmatus tadpoles, genes involved in MCC, or more Zhu et al. BMC Genomics (2018) 19:422 Page 6 of 13 Fig. 3 Expression heat map of MITF transcripts, expression level was scaled to 0–1 Fig. 4 Primary transcriptional variations in response to light exposure in O. rhodostigmatus tadpoles. a, Top 20 (sorted by q value) enriched pathways/ processes based on core light inducible gene. Rich factor is the ratio between number of gene enriched in a pathway and the total number of genes in this pathway. b, The consensus signal transduction unit among enriched pathways/processes. c, Expression heat map of genes related to epidermal growth factor, expression level was scaled to 0–1. d, Expression heat map of genes related to other growth factors, expression level was scaled to 0–1 Zhu et al. BMC Genomics (2018) 19:422 Page 7 of 13 Fig. 5 Sequence alignment of MC1R genes. Amino acids outside the frames are speculative transmembrane domains according to the deduced Human MC1R structure  exactly melanogenesis and melanocyte proliferating, in- Besides, constitutive enhancement on enzyme activities, creased with skin darkening, while those involved in facilitated by genetic mutations, go against the transpar- PCC did not (Fig. 2). Though transcriptional activation ent phenotype of dark living O. rhodostigmatus tadpoles, of related genes is not necessarily for PCC in existing whose TYR, TYRP1 and DCT genes are also transcribed, melanocytes, increased expression of these genes along even at low levels (Fig. 2a). Therefore, light-induced gene with melanocyte proliferating should be expected, if expression should be a dominate contributor to this PCC contributes to skin darkening. These results sug- rapid darkening phenomenon in these tadpoles, as sup- gested that MCC contributes to the rapid darkening of ported by the significant transcriptional upregulation of O. rhodostigmatus tadpoles. PCC in O. rhodostigmatus genes for melanin synthesis (Fig. 2a). Upstream regulat- may be not as responsible to light as it in other amphib- ing on transcription of these genes likely provides critical ians, at least in aspect of pigment dispersion. It may be insight into the potential optical switch on pigmentation resulted from long-term adaptation to darkness, where in these non-obligate cave dwellers. PCC system is less required for fine-tuning skin color. On the contrary, it is surprising that the light-induced Regulation and genetic basis of rapid skin darkening MCC of O. rhodostigmatus tadpoles is so robust to MITF is the critical transcriptional factor activating mel- darken their skin color within 15 h. Either expression or anocyte differentiation and melanogenesis in vertebrates genetic mutations of genes involved in melanin synthesis [32, 54]. The activity of MITF in melanogenesis is regu- (i.e. TYR, TYRP1 and DCT) may be responsible. lated at transcription and posttranslational modification. However, no significant functional changes on amino In O. rhodostigmatus tadpoles, the abundance of MITF acid sequences were identified in these genes of O. transcripts was as low as undetectable by our RNA-seq rhodostigmatus, in comparison with other vertebrates. programmes in lack of light, while rapid and significant Zhu et al. BMC Genomics (2018) 19:422 Page 8 of 13 Fig. 6 Sequence and structure change of MC1R and its implications in pigmentation of O. rhodostigmatus. a, Sequence analysis on the M/EJTD. Pink background indicates sites frequently occupied by basic residues. Missense mutations and amino acid deletions resulting melanic phenotypes were showed. b, Charges of this sequence fragment. c,3D model of O. rhodostigmatus MC1R, with tandem aspartates highlighted. d,Sequences and charges of major agonists of MC1R. e, Structure of α-MSH transcriptional upregulation was induced by light expos- two negatively charged amino acid residues with either ure (Fig. 3). It is interesting that light-induced tran- positively charged ones or neutral ones would reduce scription of MITF peaked after short-term exposure, the responsiveness of MC1R to α-MSH [40, 42]. Besides, different from the variation pattern of enzymes in replacement the aspartate residue at this position with melanin synthesis. It suggested that the transient lysine, asparagine, or valine decreases α-MSH binding transcriptional impulse of MITF was intensive enough affinity 10–100 fold . Similarly, replacement of 89E for initiating and maintaining melanogenesis in O. with a basic amino acid residue would also decrease rhodostigmatus tadpoles. α-MSH binding affinity . It suggested that negative Transcription of MITF is activated by cAMP signal charges in this region would reinforce the binding mediated by α-MSH (or other analogous signals) and its capacity of α-MSH. For O. rhodostigmatus, the receptor MC1R. The genetic change of MC1R should be stereo-tandem negative charges in the elongated third responsible for the rapid and intensive transcription of transmembrane helix likely performs as a ligands trap, MITF. It is known that the M/EJTD of MC1R is nega- which would enhance the ligands trapping and binding tively charged and responsible for the binding of posi- capacities of MC1R, as well as its responsiveness to li- tively changed ligands, including α-MSH and ACTH gands. It means that lower concentration of α-MSH is (Fig. 6d-e). The conserved glutamate in the second required to induce ligands-dependent activation of transmembrane helix 89E in O. rhodostigmatus MC1R) MC1R in O. rhodostigmatus than in other frogs. Besides, and the second conserved aspartate in the third trans- though it has not been reported that introducing extra membrane helix (112D in O. rhodostigmatus MC1R) are acidic amino acid residues in membrane/extracellular responsible for ligands binding, as replacement of these junction of the third transmembrane domain would Zhu et al. BMC Genomics (2018) 19:422 Page 9 of 13 improve the maximum activity of MC1R, replacement of from Yerbaniz, MC1R variant with a point mutation in its 112D with lysine, asparagine, or valine could reduce the coding region has been identified, and it resulted replace- maximum activity of MC1R. Similar outcomes were re- ment of R164 (R151 in O. rhodostigmatus)with cysteine sulted from replacement of 116C and 89E with the basic residue , which also found in humans with red hair asparagine [40, 42]. It seems that the maximum activity and pale skin . Both mutations are associated with pig- of MC1R is also related to the charge characteristic of ment regression in cave adaptive populations. They are lo- this region, and negative charges likely improve the cated out of the ligands binding region and reduce the maximum activity. Taken together, it can be speculated activity of MC1R whether constitutively or ligands de- that more efficiency and intensive activation of MC1R pendently. Besides MC1R, defects in genes involved in would be induced by α-MSH in O. rhodostigmatus than melanin biosynthesis (i.e., OCA2 and TYR) were also in other frogs, which should be a prime genetic basis for identified in cave dwellers [8, 11, 12, 34]. Obviously, these rapid MCC. types of mutations strengthen the cave adaptability at the The posttranslational modification might also play a expense of their adaptability to bright environment. role in rapid skin darkening by amplifying the MITF sig- Unlike cavefish, O. rhodostigmatus should possess nal. Phosphorylation is required for MITF activation in strong capacity of light-induced MC1R activation for MAPK pathway depended manner [24–26], and activa- lifehistory after metamorphosis, and so the mecha- tion of MAPK pathway is a common and immediate nisms underlying their cave adaptation should be dif- downstream event of GFR activation in proliferating ferent from that of cavefish. melanocyte . In O. rhodostigmatus tadpoles, light ex- Though replacement of acidic amino acid residues posure induced significant transcriptional upregulation with neutral or basic ones, or replacements of neutral of numerous growth factor related genes and core com- residues with basic ones, in membrane/extracellular ponents involved in MAPK pathway (Fig. 4). It was pos- junction of the second and third transmembrane do- sible that light-induced transcriptional activation of mains would reduce the affinity and responsiveness of growth signal and MAPK pathway was beneficial to MC1R to α-MSH, it could also enhance the constitutive MITF phosphorylation in O. rhodostigmatus tadpoles, activation of MC1R and thus results in dark phenotypes and thus facilitating rapid skin darkening. . For example, the replacement of 89E (in O. rhodos- In addition, growth signals and their downstream signal tigmatus) with asparagine was identified in the melanic pathways, typically MAPK and PI3K-Akt signal pathways, mouse , chicken and bananaquits [43, 44], the re- facilitate melanocyte proliferation in MITF-independent placement of 112D with glutamine was identified in the manner . Synergistic effect of growth factors has been melanic sheep , pig  and flycatcher , and the widely reported in melanocyte proliferation. For human replacement of 116C with asparagine was identified in melanocytes, FGFs, HGFs, M/SCGFs are strong synergis- the dark color fox . Additionally, amino acids dele- tic mitogens, α-MSH and ACTH are weak mitogens, tion in this region was also widely reported in MC1R of while EGFs and VEGFs are ineffective mitogens, and at animals with melanic phenotypes (Fig. 6a)[48–50], and least two stimulators are required for their proliferating these deletions always cover the conserved or high- . In O. rhodostigmatus melanocyte, EGFs, PDGFs, frequency sites for acidic residues. It has been proven that FGFs, HGFs and M/SCGFs related genes were synchron- positive charged residues in this region are necessary for ously upregulated, covering all the three strong synergistic constitutive activation of MC1R in manner of ligands mitogens identified for human melanocytes. Therefore, a mimicking [40, 42]. Accordingly, it can be speculated that strong proliferative stimulus can be expected from their introducing extra acidic residues, or replacements of basic synergistic effects. The strong synergistic effect of growth residues with neutral and acidic ones, which is exactly the signal may be one of the reasons for the rapid skin dark- situation in O. rhodostigmatus, would weaken the consti- ening, if it is proved to be associated with melanocyte pro- tutive activity of MC1R. It provided a genetic explanation liferation in O. rhodostigmatus. for the transparent phenotype of O. rhodostigmatus tad- poles. It also explained why permanent pigment regression Genetic basis of pigment regression or pale phenotypes granted by acidifying the M/EJTD of In contrast to rapid MCC, cave adaptation requires re- MC1R has not been identified in animals [9, 51, 52], even duced MC1R activity to keep their transparent skin melanistic phenotypes granted by basic residues introdu- color. Astyanax mexicanus is the most studied cavefish, cing and/or acid residues eliminating in these regions are whose cave population showed obvious pigment regres- common in vertebrates [40–50]. sion in comparison with surface populations. In cave Overall, the fragment deletion and extra acidic amino population collected from Pachon, MC1R variant with acid residues in the ligands binding domain of MC1R 2bpdeletionin coding regionresulted in non-functional likely reduced the constitutive activity of MC1R, but re- product has been identified . While in cave population inforced its ligands-dependent activity, which might Zhu et al. BMC Genomics (2018) 19:422 Page 10 of 13 contribute to pigment regression for cave adaptation requirements. To our knowledge, this is the first study and rapid MCC for transforming of life history, re- that reported the association between pigmentation spectively (Fig. 7). phenotype adaptation and MC1R mutations in amphib- ians and/or in the non-obligate cave dwellers. Conclusion Tadpoles of O. rhodostigmatus are non-obligate cave Methods dwellers, who keep transparent phenotype in caves but Sample collection and treatment rapidly darken in light within 15 h. Using comparative Tadpoles of Oreolalax rhodostigmatus were collected in a transcriptomics, we found that the melanocyte MCC (in- karst cave of Shizhu County, Chongqing City, China in cluding melanogenesis and melanocytes proliferation) April 2017. In the cave, a stream runs from inside to out- was responsible for the rapid skin darkening in O. rho- side, with stable water temperature of 15.3 °C. When dostigmatus tadpoles. As the most prominent change, Beijing time is five a.m., a total of 13 “transparent” tadpoles light-induced transcriptional activation of growth signals were collected simultaneously from a pool in the dark zone (including growth factor signals, MAPK signal pathways of the cave, and all of them were identified at their Gosner and PI3K-Akt signal pathways) may facilitate the rapid stage of 25 . Once collected, four of them were immedi- MCC in O. rhodostigmatus tadpoles. The most amazing ately anaesthetized and sacrificed to collect eyeballs, liver 3 2 found here is that an in-frame deletion of four amino (0.5 cm ), dorsal skin (1 cm ) and half tail (3 cm length). acids in the M/EJTD of O. rhodostigmatus MC1R, the Tissues of each tadpole were all together placed into one receptor for melanogenesis signal, was identified. This tissue tube as one sample, and then preserved in liquid ni- mutation increases the negative charge of the ligand trogen. These four samples were defined in a “Control pocket of MC1R and results in the stereo-tandem of group”. The rest tadpoles were raised in the cage immersed three aspartate residues aligning towards its ligand in the pool at the entrance of the cave, where is pocket. The ligand pocket of O. rhodostigmatus MC1R light-accessible, and after 8 p.m. a broad-spectrum fluores- resembles a trap for positively charged ligands (α-MSH cent lamp was used to imitate sunlight at night. Four hours and ACTH) and likely increases the ligands-dependent later, six tadpoles were anaesthetized and sacrificed to col- activity of MC1R, providing an explanation for the rapid lect tissues as described above which were defined as a MCC of O. rhodostigmatus in light. Meanwhile, in- “Short-term exposed group”, and tissues of each tadpole creased negative charge of ligand pocket likely decreased were all together placed into one tissue tube as one sample. the constitutive activity of MC1R, supporting the trans- The rest three tadpoles were similarly collected after 30 h parent phenotype of the cave-dwelling tadpoles. There- of light exposure, and these samples were defined as a fore, genetic mutations of MC1R explains, at least to “Long-term exposed group”. some extent, how the pigmentation system of O. rhodos- tigmatus coordinates the capacity of rapid melanogenesis cDNA library construction and Illumina sequencing (or other types of pigment production) and pigment re- Total RNA of each sample was extracted and purified gression, a couple of seemingly contradictory coloration using Trizol (Invitrogen, Carlsbad, CA, USA) following Fig. 7 Deduced mechanism by which O. rhodostigmatus MC1R coordinates pigment regression in darkness and rapid melanogenesis (or other types of pigment production) in response to light exposure Zhu et al. BMC Genomics (2018) 19:422 Page 11 of 13 the manufacturer’s instructions. After purified with Analysis of differentially expressed genes poly-T oligo-attached magnetic beads, the mRNAs were The analysis flow was showed in Fig. 1 H. Briefly, uni- fragmented. First-strand cDNA was synthesized using genes, whose expression levels showed upregulation with random hexamer primers and M-MuLV Reverse Tran- light exposure (false discovery rate < 0.05, one-way scriptase (RNase H−). Second-strand cDNA synthesis ANOVA & BHfdr), were considered to be light inducible was subsequently performed using DNA Polymerase I genes. On this basis, those showed significant upregula- and RNase H. The remaining overhangs were converted tions in pairwise comparisons, “short-term exposed vs into blunt ends via exonuclease/polymerase activities. control” and “long-term exposed vs short-term exposed”, After adenylation of the 3′ ends of the DNA fragments, were stricter light inducible genes. These unigenes were NEBNext adaptors with a hairpin loop structure were uploaded to Kobas 3.0 (http://kobas.cbi.pku.edu.cn/ ligated to prepare for hybridization. To preferentially se- index.php) for enrichment analysis . Information of lect cDNA fragments of 150–200 bp in length, the unigenes used in analysis and figures was summarized in library fragments were purified with AMPure XP system Additional file 8: Table S6. (Beckman Coulter, Beverly, USA). Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, Sequence comparison and phylogenetic analyses adaptor-ligated cDNA at 37 °C for 15 min followed by Sequences of targeted genes were retrieved from Genbank 5 min at 95 °C before PCR. Then, PCR was performed or our transcriptome database. N-J tree was built on with a Phusion High-Fidelity DNA polymerase, universal MEGA7 with default parameters. Sequence alignment was PCR primers and Index (X) primer. Finally, PCR prod- performed on Clustal X2, and further edit was performed ucts were purified (AMPure XP system), and library on GeneDoc. quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was Prediction of protein 3D models performed on a cBot Cluster Generation System using 3D models of MC1R proteins were predicted on the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) ac- Swiss-model server (https://www.swissmodel.expasy.org/) cording to the manufacturer’s instructions. After cluster with “Chimera protein of human 5-hydroxytryptamine re- generation, the library preparations were sequenced on ceptor 1B (ID: 4iaq.1)” as model . Analysis of 3D an Illumina HiSeq 4000 platform by NovoGene (Beijing), models was performed on Swiss PDB Viewer. and paired-end reads were generated. Analysis of peptide property Peptide physicochemical property was analyzed on Pep- De novo transcriptome assembly and transcriptome Draw online server (http://www.tulane.edu/~biochem/ annotation WW/PepDraw/index.html). The reads quality was verified using FastQC (version 0.10.0) software. Reads containing adapters, reads con- Additional files taining poly-N and low-quality reads were removed. The total clean reads from all libraries were assembled de Additional file 1: Table S1. Summary of sequencing quality. (XLSX 9 kb) novo using Trinity as a reference transcriptome. The re- Additional file 2: Figure S1. Length distribution of transcripts and sulted unigenes were annotated by querying databases unigenes. (PDF 4 kb) NR, NT and Swiss-Prot with an E-value threshold of Additional file 3: Table S2. Unigene annotation details. (XLSX 49576 kb) 1.0E-5 and KOG with an E-value threshold of 1.0E-3. Additional file 4: Table S3. Summary of FPKM. (XLSX 20703 kb) Then, Blast2GO software was used to obtain GO annota- Additional file 5: Table S4. Summary of enrichment based on light- tions defined by molecular function, cellular component inducible genes (1662). (XLSX 46 kb) and biological process ontologies. Pathway assignments Additional file 6: Table S5. Summary of enrichment based on stricter light-inducible genes (213). (XLSX 32 kb) were determined based on the KEGG database using Additional file 7: Figure S2. 3D-model of MC1R in frogs. Models are BLASTX with an E-value threshold of 1.0E-5. built based on human 5-hydroxytryptamine receptor. Red arrows indicate ending point of TMH3 in extracellular side. (PDF 11742 kb) Additional file 8: Table S6. Information of unigenes used in analysis Validity assessment of transcriptome data and figures. (XLSX 538 kb) The expression level of each unigene was expressed as fragments per kilobase of exon model per million Abbreviations mapped reads (FPKM). Pearson correlations of gene ex- ACTH: Adrenocorticotropic hormone; ASIP: Agouti signal peptide; BCO1: Beta, pression levels between samples were calculated to beta-carotene 15,15′-monooxygenase; BCO2: Beta, beta-carotene 9′,10′- oxygenase; DCT: Dopachrome tautomerase; DHPR: Dihydropteridine assess the validity of transcriptome data. Generally reductase; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; speaking, valid data should have higher intra-group cor- GCH1: GTP cyclohydrolase 1; GCHFR: GTP cyclohydrolase 1 feedback relations than inter-group ones. regulatory protein; GFR: Growth factor receptor; HGF: Hepatocyte growth Zhu et al. BMC Genomics (2018) 19:422 Page 12 of 13 factor; M/SCGF: Mast/stem cell growth factor; MC1R: Melanocortin-1 7. Jeffery WR, Li M, Parkhurst A, Bilandžija H: Pigment regression and albinism receptor; MCC: Morphological color change; MCH: Melanin-concentrating in Astyanax cavefish. 2014. hormone; MCHR: MCH receptor; MELANA: Melanoma antigen recognized 8. Yang J, Chen X, Bai J, Fang D, Qiu Y, Jiang W, Yuan H, Bian C, Lu J, He S. by T-cells 1 isoform X1; MITF: Microphthalmia-associated transcription factor; The Sinocyclocheilus cavefish genome provides insights into cave adaptation. PCC: Physiological color change; PDGF: Platelet-derived growth factor; BMC Biol. 2016;14(1):1. PMEL: Premelanosome protein precursor; POMC: Precursor proopiomelanocortin 9. Gross JB, Borowsky R, Tabin CJ. A novel role for Mc1r in the parallel evolution protein; SPR: Sepiapterin reductase; TYR: Tyrosinase; TYRP1: Tyrosinase-related of depigmentation in independent populations of the cavefish Astyanax protein 1; XDH/XOD: Xanthine dehydrogenase/oxidase; α-MSH: alpha-Melanocyte- mexicanus. PLoS Genet. 2009;5(1):e1000326. stimulating hormone 10. Protas M, Jeffery WR. Evolution and development in cave animals: from fish to crustaceans. Wiley Interdiscip Rev Dev Biol. 2012;1(6):823–45. Acknowledgements 11. McCauley DW, Hixon E, Jeffery WR. Evolution of pigment cell regression in We thank the editors and reviewers for their work on promoting the manuscript. the cavefish Astyanax: a late step in melanogenesis. Evolution & Development. We thank Dengwei Yang for his help on collecting O. rhodostigmatus tadpoles. 2004;6(4):209–18. 12. Protas ME, Hersey C, Kochanek D, Zhou Y, Wilkens H, Jeffery WR, Zon LI, Funding Borowsky R, Tabin CJ. Genetic analysis of cavefish reveals molecular This work was supported by National Natural Sciences Foundation of China convergence in the evolution of albinism. Nat Genet. 2006;38(1):107–11. (NSFC-31201702 and NSFC-31471964), National Key Programme of Research 13. Bilandžija H, Ma L, Parkhurst A, Jeffery WR. A potential benefit of albinism in and Development, Ministry of Science and Technology (2017YFC0505202), Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and the Strategic Priority Research Program of the Chinese Academy of Sciences catecholamine levels as an alternative to melanin synthesis. PLoS One. 2013; (XDA19050201 and XDPB0202), Important Research Project of Chinese Academy 8(11):e80823. of Sciences (KJZG-EW-L13) and 2017 Western Light Talent Culture Project of the 14. Fei L, Ye C, Jiang J. Colored atlas of Chinese amphibians and their distributions. 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Published: May 31, 2018
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