Yi H et al. (2025), Melanin deposition and key molecular features i...

XB-ART-61267

BMC Biol 2025 Feb 27;231:62. doi: 10.1186/s12915-025-02168-0.

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Melanin deposition and key molecular features in Xenopus tropicalis oocytes.

Yi H , Liang W , Yang S , Liu H , Deng J , Han S , Feng X , Cheng W , Chen Y , Hang J , Lu H , Ran R .

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BACKGROUND: Melanin pigmentation in oocytes is a critical feature for both the esthetic and developmental aspects of oocytes, influencing their polarity and overall development. Despite substantial knowledge of melanogenesis in melanocytes and retinal pigment epithelium cells, the molecular mechanisms underlying oocyte melanogenesis remain largely unknown. RESULTS: Here, we compare the oocytes of wild-type, tyr-/- and mitf-/- Xenopus tropicalis and found that mitf-/- oocytes exhibit normal melanin deposition at the animal pole, whereas tyr-/- oocytes show no melanin deposition at this site. Transmission electron microscopy confirmed that melanogenesis in mitf-/- oocytes proceeds normally, similar to wild-type oocytes. Transcriptomic analysis revealed that mitf-/- oocytes still express melanogenesis-related genes, enabling them to complete melanogenesis. Additionally, in Xenopus tropicalis oocytes, the expression of the MiT subfamily factor tfe3 is relatively high, while tfeb, mitf, and tfec levels are extremely low. The expression pattern of tfe3 is similar to that of tyr and other melanogenesis-related genes. Thus, melanogenesis in Xenopus tropicalis oocytes is independent of Mitf and may be regulated by other MiT subfamily factors such as Tfe3, which control the expression of genes like tyr, dct, and tyrp1. Furthermore, transcriptomic data revealed that changes in the expression of genes related to mitochondrial cloud formation represent the most significant molecular changes during oocyte development. CONCLUSIONS: Overall, these findings suggest that further elucidation of Tyr-dependent and Mitf-independent mechanisms of melanin deposition at the animal pole will enhance our understanding of melanogenesis and Oogenesis.

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Species referenced: Xenopus tropicalis
Genes referenced: atp7a best1 dapl1 dct ednrb2 gpnmb kcnj13 mapk1 mc1r mfn1 mfn2 mitf mlana mlph msi2 nf2 nrf1 opa1 pmel ppargc1a rdh5 rlbp1 rpe65 serpinf1 slc24a5 slc45a2 tacc3 tfam tfe3 tfeb tfec trpm1 tyr tyrp1 zp3 zp3.2 zp4 zp4.2
GO keywords: Wnt signaling pathway

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	Fig. 1. The mitf−/−Xenopus tropicalis. A The knockout site of mitf in Xenopus tropicalis. B The genotype of mitf−/− Xenopus tropicalis (n = 40 tadpoles). C Dorsal and ventral views of representative wild-type (WT) and mitf−/− Xenopus tropicalis (WT, n = 10 froglets; mitf−/−, n = 10 froglets). D Dorsal and ventral views of representative WT and mitf−/− Xenopus tropicalis at 1 year old (WT, n = 10 frogs; mitf−/−, n = 10 frogs). E Hematoxylin and eosin staining of representative dorsal skin tissue sections from WT, mitf−/−, and mitf−/− rescue (G0 generation after gene knockout site repair) Xenopus tropicalis. Three frogs of each genotype were examined. Tissue samples were embedded in paraffin and sectioned at 6 μm thickness, with at least 10 paraffin sections observed per frog. Black arrows indicate melanocytes. F Hematoxylin and eosin staining of representative eye tissue sections from WT and mitf−/− Xenopus tropicalis. Three frogs of each genotype were examined. Tissue samples were embedded in paraffin and sectioned at 6 μm thickness, with at least 10 paraffin sections observed per frog. Black arrows indicate the RPE, and red arrows indicate melanocytes in the choroid layer. DS, dorsal view; VS, ventral view. Scale bars: 5 mm in C and D, 50 μm in E and F
	Fig. 2. Xenopus tropicalis Mitf regulates the expression of genes related to melanocyte development. A The cartoon provides a schematic representation of how the human MITF protein regulates the expression of target genes via the M-box (CACGTG), thereby controlling melanocyte development. B, C Heat maps show the differential expression of genes associated with melanocyte development in the dorsal (B) and ventral (C) skin of WT and mitf−/− Xenopus tropicalis. The FPKM values for each gene were normalized using the normalization function in GraphPad Prism 8.0 software, which was also used to create the heat maps. Mcon-DS1, Mcon-DS2, MKO-DS1, and MKO-DS2 represent two replicates of dorsal skin from WT and mitf−/− Xenopus tropicalis, respectively. Similarly, Mcon-VS1, Mcon-VS2, MKO-VS1, and MKO-VS2 represent two replicates of ventral skin from WT and mitf−/− Xenopus tropicalis, respectively. Each replicate sample from wild-type and mitf−/− Xenopus tropicalis was obtained from the dorsal or ventral skin of three frogs. D–K The expression of Mitf-activated target genes (rdh5, rlbp1, msi1, dapl1, nf2, best1, ppargc1a, serpinf1) in the eyes of WT and mitf−/− Xenopus tropicalis (unpaired t test)
	Fig. 3. Tyr regulates melanin deposition in Xenopus tropicalis oocytes. A–C Representative images show the ovarian from 1-year-old WT, mitf−/−, and tyr−/− female Xenopus tropicalis, with three frogs observed for each genotype (n = 9 frogs). In A and B, magnified views of black spots on the ovarian peritoneum, indicated by blue arrows in the upper panels, are shown in the corresponding lower panels and are also indicated by blue arrows. The scale bars in the upper panels of A, B, and C are 0.5 mm, and in the lower panels, they are 0.1 mm. D, F Figures present TEM images of oocytes from one-year-old WT and mitf−/− Xenopus tropicalis. E and G are enlarged views of the regions indicated by red arrows in D and F, respectively. H, J Figures display TEM images of black spots on the ovarian peritoneum of 1-year-old WT and mitf−/− Xenopus tropicalis. I and K are enlarged views of the regions indicated by red arrows in H and J, respectively. The scale bars are as shown in the images. All images in D–K are representative (n = 3 frogs). L–O Figures show the mRNA expression levels of mitf, tyr, tfe3, and tfec during oocyte development in WT and mitf−/− Xenopus tropicalis, presented as FPKM values. P The cartoon provides a schematic diagram summarizing the possible molecular mechanisms regulating melanin deposition in Xenopus tropicalis oocytes. For the P values of the data in L–O, please refer to Supplementary Table S3. The statistical tests are the Wald test and the Likelihood ratio test in Deseq2
	Fig. 4. MiT family transcription factors Tfe3, Mitf, and Tfec regulate the expression of the tyr and dct genes. A–B show the nucleotide frequency of different bases at various positions within the DNA binding motifs for Mitf and Tfe3. Data were obtained from JASPAR. C The DNA binding motifs for Mitf and Tfe3 in the promoter region of tyr and dct (2000 bp upstream of the transcription start site, TSS). The blue numbers indicate the distance (in base pairs) from the TSS. Data were obtained from JASPAR and NCBI. D–E Present dual-luciferase assay results for the regulation of tyr and dct expression by Mitf, Tfe3, and Tfec (n = 4). *** indicates a P value < 0.001, as determined by an unpaired t test
	Fig. 5. The expression of key genes involved in pigment synthesis progressively decreases during oocyte development in Xenopus tropicalis. A During oocyte development in WT and mitf−/− Xenopus tropicalis, the mRNA expression levels of genes associated with melanin synthesis pathways are shown as FPKM values. B, C During oocyte development in WT and mitf−/− Xenopus tropicalis, the mRNA expression levels of genes associated with carotenoid and pteridine synthesis pathways are shown as FPKM values. For the P values of the data in figures, please refer to Additional file 2: Table S3. The statistical tests are the Wald test and the Likelihood ratio test in Deseq2
	Fig. 6. Differentially expressed genes during oogenesis development in Xenopus tropicalis. A Expression of mitochondria-related genes during oogenesis in WT Xenopus tropicalis. B Expression of mitochondria-related genes during oogenesis in mitf−/− Xenopus tropicalis. C Expression of key genes regulating mitochondrial biogenesis during oogenesis in WT Xenopus tropicalis. D Expression of key genes regulating mitochondrial biogenesis during oogenesis in mitf−/− Xenopus tropicalis. E Expression of zona pellucida formation-related genes during oogenesis in WT Xenopus tropicalis. F Expression of zona pellucida formation-related genes during oogenesis in mitf−/− Xenopus tropicalis. G–J Average expression of zona pellucida formation-related genes zp2, zp3.2, zp4.2, and zp4 during oogenesis in WT and mitf−/− Xenopus tropicalis. K–L Average expression of meiosis-related genes pcnt and tacc3 during oogenesis in WT and mitf−/− Xenopus tropicalis. Results are shown as FPKM values. For A–F, the statistical tests are the Wald test and the Likelihood ratio test in Deseq2. For G–L, the statistical tests are the one-way ANOVA
	Supplementary Figure S1. Transcripts of human MITF (A) and mouse Mitf (B). For detailed information, refer to NCBI
	Supplementary Figure S2. In mitf-/- Xenopus tropicalis, the bHLHzip domain of the Mitf protein, is inactivated. A, the picture provides a schematic representation of the transcripts and related information of the mitfa gene locus in zebrafish and the mitf gene locus in Xenopus tropicalis. The red arrow indicates the penultimate exon, which is responsible for transcribing a common part of the transcripts from this gene locus. B and C, pictures display two different views of the 3D structure of the human MITF protein (encoded by transcript variant 4 of the human MITF gene locus, a master regulator of melanocyte development). The yellow-highlighted regions show the conserved amino acid sequences between the Xenopus tropicalis Mitf protein and the human MITF protein. The red arrow points to the knockout site in the mitf-/- Xenopus tropicalis Mitf protein. 'N' indicates the amino-terminal, and 'C' indicates the carboxy-terminal. D and E, the figure illustrate the amino acid sequence alignment of feature 1 (DNA binding site) and feature 2 (polypeptide binding site/dimer interface) of the bHLHzip domain among different vertebrate species. In D and E, red indicates high conservation and blue indicates low conservation. Hash-marks (#) in the top row of the multiple sequence alignment display indicate specific residues involved in a conserved feature, such as a binding or catalytic site, that has been annotated on an NCBI-curated domain. The black arrows in A, D, and E indicate the knockout site of mitf-/- Xenopus tropicalis mitf. For detailed data analysis, refer to NCBI (https://www.ncbi.nlm.nih.gov/)
	Supplementary Figure S3. The transcriptomic comparison of dorsal (DS) and ventral (VS) skin samples from wild-type Xenopus tropicalis revealed significant overall differences in the expression of melanogenesis-related genes (P = 0.0139, paired t-test).
	Supplementary Figure S4. Differential expression of genes related to RPE cells and melanocytes in the eyes of WT and mitf-/- Xenopus tropicalis. Mcon denotes WT Xenopus tropicalis eye samples, while MKO denotes mitf-/- Xenopus tropicalis eye samples.
	Supplementary Figure S6. A-C, Expression levels of tfeb,tyrp1, and dct mRNA during oocyte development in WT and mitf-/- Xenopus tropicalis. D, Comparison of amino acid sequences of human MITF-A, Xenopus tropicalis Mitf, and Xenopus tropicalis Tfe3 proteins. Protein sequence information was obtained from https://www.uniprot.org/.
	Supplementary Figure S7. The DNA motifs bound by MiT family transcription factors were derived from JASPAR (http://jaspar.genereg.net/). Panels A-B, C-D, E-F, and G-H display the nucleotide frequency and motif sequences at different positions for Tfe3, Mitf, Tfeb, and Tfec, respectively.
	Supplementary Figure S8. The dorsal views of adult WT, mitf-knockout (mitf-/-), and mitf-/- rescue Xenopus tropicalis were compared. At least 100 frogs were analyzed for each group, except for the mitf-/- rescue. The scale bar represents 5 mm
	Supplementary Figure S9 The homology of zona pellucida proteins between Xenopus tropicalis and human oocytes was compared using data from the UniProt database.
	Supplementary Figure S10 The homology of zona pellucida proteins between Xenopus tropicalis and human oocytes was compared using data from the UniProt database.
	Supplementary Figure S11 The homology of zona pellucida proteins between Xenopus tropicalis and human oocytes was compared using data from the UniProt database.
	Supplementary Figure S12 The homology of zona pellucida proteins between Xenopus tropicalis and human oocytes was compared using data from the UniProt database.