Dispensable role of mitf in melanogenesis of Xenopus tropicalis oocytes

Dispensable role of mitf in melanogenesis of Xenopus tropicalis oocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dispensable role of mitf in melanogenesis of Xenopus tropicalis oocytes Hongyang Yi, Jing Hang, Jiayin Shen, Sumei Yang, Han Liu, Jiayu Deng, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4807093/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Melanin pigmentation in oocytes is a critical feature for both the aesthetic 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. 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 regulate the expression of melanogenesis-related genes 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 dependent on Tyr rather than Mitf, possibly due to the regulation of tyr , dct , and tyrp1 by other MiT subfamily factors such as tfe3 . 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. Overall, these findings suggest that further elucidation of Tyr-dependent, Mitf-independent mechanisms of melanin deposition at the animal pole will enhance our understanding of melanogenesis and Oogenesis. Oogenesis melanogenesis Xenopus tropicalis mitochondrial cloud Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Oogenesis, the process by which oocytes (egg cells) develop, involves complex interactions among genetic, biochemical, and environmental factors( 1 ). In some species, oocytes undergo melanin deposition during this process. Melanin pigmentation in oocytes is a critical feature that affects not only the aesthetic attributes of the eggs but also their survival and fitness in natural environments( 2 – 5 ). For instance, melanin pigmentation can protect the developing oocyte from UV radiation and oxidative stress, thereby contributing to the overall viability of the embryo( 6 , 7 ). Additionally, melanin pigment granules may play a role in the thermal regulation of the eggs, influencing embryonic development rates( 8 ). Therefore, understanding the molecular mechanisms governing melanin synthesis in oocytes provides insights into oocyte development and other broader biological phenomena, such as gene regulatory networks, organelle biogenesis, cellular differentiation, and intracellular transport. In vertebrates, melanogenesis primarily occurs in melanocytes, the retinal pigment epithelium (RPE), and the oocytes of certain species( 9 , 10 ). The melanogenesis process involves a series of enzymatic reactions that begin with the amino acid tyrosine( 11 , 12 ). Central to melanogenesis is the enzyme tyrosinase, which catalyzes the initial and rate-limiting steps of melanin synthesis, converting tyrosine into dihydroxyphenylalanine (DOPA) and subsequently into dopaquinone( 9 , 11 – 13 ). Further complex biochemical transformations lead to the production of different types of melanin, mainly eumelanin (brown to black pigment) and pheomelanin (yellow to red pigment)( 11 ). The type and amount of melanin produced are regulated by genetic, hormonal, and environmental factors, making melanogenesis a highly dynamic and tightly controlled process. The molecular regulation of melanogenesis is predominantly controlled by the microphthalmia-associated transcription factor (MITF), a master regulator in melanocytes that governs the expression of essential melanogenic enzymes and structural proteins( 11 , 12 , 14 ). MITF specifically regulates enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2)( 11 , 14 ). MITF activity is modulated by several signaling pathways, including cAMP/protein kinase A (PKA), Wnt/β-catenin, and mitogen-activated protein kinase (MAPK)( 13 , 14 ). These pathways are activated by various extracellular signals, such as UV radiation, hormones, and cytokines, which collectively modulate MITF expression and activity, highlighting the complexity of melanin production regulation. Despite significant advances in understanding the molecular mechanisms of melanogenesis( 14 , 15 ), many aspects remain unclear. The roles of non-coding RNAs, epigenetic modifications, and genetic polymorphisms in melanogenic pathways are areas of ongoing research( 16 , 17 ). It is noteworthy that although melanogenesis plays a crucial role in Xenopus oocyte development, the molecular regulatory mechanisms of Xenopus oocyte melanogenesis remain largely unknown( 4 , 5 , 18 – 24 ). Previous research has shown that tyrosinase activity is markedly higher in stage III and stage IV Xenopus oocytes compared to other stages( 20 , 24 ). Additionally, early studies have found that tyrosinase activity is present in both albino and wild-type Xenopus oocytes, with albino oocytes exhibiting higher enzyme activity( 22 ). Numerous experiments have confirmed that the tyrosinase inhibitor PTU can induce the production of melanin-free oocytes and tadpoles in Xenopus ( 5 , 19 , 24 ). Furthermore, gene-editing technologies such as TALEN and CRISPR/Cas9 have produced tyrosinase knockout lines of Xenopus laevis and Xenopus tropicalis ( 25 , 26 ). However, the molecular mechanisms regulating tyrosinase expression in oocytes remain unclear. Due to the significant differences between the intracellular and extracellular environments of oocytes and melanocytes( 1 , 15 , 27 , 28 ), the similarities and differences in the molecular regulatory mechanisms of melanogenesis in oocytes and melanocytes are still unknown. In our previous research, we successfully established mitf −/− and tyr −/− Xenopus tropicalis lines using CRISPR/Cas9 gene editing technology( 26 ). We discovered that while tyr −/− Xenopus tropicalis oocytes completely lacked melanin deposition, mitf −/− Xenopus tropicalis oocytes exhibited normal melanin deposition( 26 ). This unexpected oocyte phenotype further motivated us to study the molecular regulatory mechanisms of oocyte melanogenesis in Xenopus tropicalis . Therefore, in this study, we utilized oocytes from three genotypes of Xenopus tropicalis : WT, mitf −/− , and tyr −/− , to investigate the molecular characteristics of oocyte melanogenesis. Our results revealed that although the core enzymes involved in Xenopus tropicalis oocyte melanogenesis are tyrosinase, tyrosinase-related protein 1 (Trp1), and tyrosinase-related protein 2 (Trp2), the master regulator of these core enzymes' expression is not Mitf. Thus, our findings suggest that Xenopus tropicalis oocyte melanogenesis depends on a master regulator other than Mitf, revising our understanding of the function of the mitf gene and highlighting the importance of further exploring the molecular regulatory mechanisms of oocyte melanogenesis. Results Disruption of the Mitf basic helix-loop-helix leucine zipper domain impairs its function Microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family, is highly conserved and serves as a pivotal regulator in numerous biological processes, including cellular differentiation, proliferation, and survival across various tissues( 14 , 29 ). Initially identified for its role in ocular development, MITF has since emerged as a multifaceted transcription factor with implications extending far beyond its namesake. Its intricate involvement in melanocyte biology, osteoclastogenesis, and immune response underscores its versatile functionality( 14 , 30 , 31 ). Structurally, MITF exhibits a modular organization characterized by distinct domains responsible for DNA binding (the bHLH-LZ domain binds DNA as dimers), protein-protein interactions, and transcriptional activation (a strong transcription activation domain at the N-terminus and a much weaker second transactivation domain at the C-terminus) ( 14 , 29 ). These domains intricately cooperate to confer specificity in target gene recognition, recruitment of cofactors, and modulation of transcriptional activity( 29 ). When both transcription activation domains (TADs) of MITF are inactivated, the mutated MITF can form dimers with wild-type MITF, exerting a dominant negative effect( 32 ). Given that the bHLH-LZ domain is primarily responsible for the DNA-binding function of MITF, disrupting this domain results in the loss of MITF function( 14 , 29 ). Therefore, we utilized CRISPR/Cas9 gene editing technology to disrupt the bHLH-LZ domain of Mitf in Xenopus tropicalis to achieve mitf gene knockout( 26 ). Generally, the transcription of the MITF gene locus mRNA in mammals is complex and variable (Supplementary Figure S1 ). However, NCBI records show only one mitf transcript for Xenopus tropicalis and three mitf transcripts for zebrafish (Figure S2 .A). This discrepancy may be due to limited research on mitf mRNA transcription in these species. Analysis of MITF mRNA transcription in humans, mice, Xenopus tropicalis , and zebrafish reveals variability at the 5' end and consistency at the 3' end (Supplementary Figure S1 and Figure S2 .A). Within the same species, the final few exons encoding these isoforms are identical, resulting in all MITF protein isoforms having the same C-terminus, likely due to the presence of highly conserved bHLH-LZ and TAD domains in these regions( 14 , 29 ). To disrupt all isoforms encoded by the Xenopus tropicalis mitf gene locus, we designed a Cas9-targeted knockout site on the penultimate exon (Fig. 1 .A and Figure S2 .A-C). According to Alphafold predictions, the bHLH-LZ and TAD domains are highly conserved between Xenopus tropicalis Mitf and human MITF (Figure S2 .B-C). The DNA and polypeptide binding sites within the bHLH-LZ domain exhibit significant conservation across species, and our designed guide RNA targets these conserved sites (Figure S2 .D-E). This suggests that in mitf −/− Xenopus tropicalis , the core bHLH-LZ domain of Mitf isoforms is disrupted, impairing their function. Indeed, following CRISPR/Cas9 knockout of mitf in Xenopus tropicalis , the resulting mitf −/− frogs lack melanocytes, xanthophores, and granular glands (Fig. 1 .B-F)( 26 ). During vertebrate eye development, various Mitf isoforms play crucial regulatory roles. Studies on various Mitf mutant mouse strains demonstrate that mutations in the bHLH-LZ domain of mouse MITF lead to abnormal eye development, including smaller eyes and aberrant pigmentation( 30 ). In contrast, mitf −/− Xenopus tropicalis exhibit normal eye size with apparently normal eye pigmentation( 26 ). Given the complexity of MITF's regulatory role in eye development and the subtle nature of some phenotypic abnormalities( 30 ), further research is needed to assess whether the retina and RPE development in mitf −/− Xenopus tropicalis are truly unaffected (Fig. 1 .F). Overall, these data suggest that in mitf −/− Xenopus tropicalis , the disruption of the core bHLH-LZ domain of Mitf impairs the critical regulatory functions of various Mitf isoforms, particularly in melanocyte development and melanogenesis. Absence of melanogenesis in the skin of mitf −/− Xenopus tropicalis To investigate the molecular changes in Mitf-regulated target gene expression in mitf knockout Xenopus tropicalis , we performed Bulk RNA-seq on dorsal and ventral skin samples from adult wild-type (WT) and mitf −/− frogs. It is well-established that genes involved in melanogenic enzymes (e.g., TYR , TYRP1 , DCT ), melanosome ionic equilibrium (e.g., SLC45A2 , SLC24A5 ), melanocyte development signaling pathways (e.g., MC1R ), and melanosome biogenesis (e.g., MLANA , PMEL ) are regulated by MITF (Fig. 2 .A). We first assessed the expression of these melanogenesis-related target genes. As expected, the expression levels of tyr , tyrp1 , dct , slc45a2 , slc24a5 , mc1r , mlana , and pmel were significantly reduced in both dorsal and ventral skin samples of mitf −/− Xenopus tropicalis (Fig. 2 .B-C and Supplementary Table S1 ). Additionally, melanocyte marker genes such as slc24a4 , ednrb2 , kcnj13 , trpm1 , and mlph also exhibited significantly reduced expression in the skin samples of mitf −/− frogs (Fig. 2 .B-C and Supplementary Table S1 ). We then examined the expression of these genes in eye samples from adult WT and mitf −/− Xenopus tropicalis . However, the expression of melanogenesis-related genes was not consistently reduced in the eyes of mitf −/− frogs (Supplementary Table S1 ). This discrepancy may be attributed to the presence of RPE cells in the retina, despite the absence of melanocytes in the choroid of mitf −/− Xenopus tropicalis eyes( 26 ). In mammals, MITF significantly influences the development of the retinal pigment epithelium (RPE) and retina by regulating the expression of genes such as RDH5 , RLBP1 , MSI2 , DAPL1 , miR204/211, NF2 , BEST1 , PGC1α , PEDF ( SERPINF1 ), PMEL , TYRP1 , and TYR ( 30 ). However, in mitf −/− Xenopus tropicalis eyes, the expression levels of rdh5 , rlbp1 , msi2 , dapl1 , miR204/211, nf2 , best1 , pgc1α , and pedf did not show significant changes compared to wild-type eyes (Fig. 2 .D-K and Supplementary Table S2 ). Similarly, the expression of melanogenesis-related genes such as pmel , tyrp1 , and tyr , which are regulated by MITF, were not significantly decreased in mitf −/− Xenopus tropicalis eyes (Supplementary Figure S3 and Supplementary Table S2 ). Additionally, there was no significant difference in the expression of rpe65 , a gene crucial for RPE cells, between wild-type and mitf −/− Xenopus tropicalis eyes (Supplementary Figure S3 and Supplementary Table S2 ). These findings indicate that RPE cells in mitf −/− Xenopus tropicalis eyes can still develop into functional RPE cell layers despite the disruption of the Mitf bHLH-LZ domain( 26 ). However, the absence of melanocytes in the choroid layer of mitf −/− Xenopus tropicalis eyes confirms the lack of melanocyte development and melanogenesis( 26 ). In conclusion, this molecular evidence demonstrates that disrupting the Mitf bHLH-LZ domain in Xenopus tropicalis leads to a loss of Mitf activity, resulting in the absence of melanocytes and melanogenesis in the skin samples of mitf −/− Xenopus tropicalis , thereby contributing to their colorless and transparent appearance( 26 ). Normal oocyte melanin deposition in mitf −/− Xenopus tropicalis during oogenesis In mitf −/− Xenopus tropicalis , the absence of melanocyte development in the skin led us to predict that oocytes in female mitf −/− Xenopus tropicalis would also lack melanin deposition( 26 ). However, contrary to our prediction, melanin deposition in mitf −/− Xenopus tropicalis oocytes was normal, with their pigmentation pattern being almost indistinguishable from that of wild-type oocytes (Fig. 3 .A-B) ( 26 ). Additionally, the ovarian membrane of mitf −/− Xenopus tropicalis , which encases the ovaries, exhibited black spots similar to those observed in wild-type ovarian membranes (Fig. 3 .A-B). In contrast, tyr −/− Xenopus tropicalis lacked black spots on the ovarian membrane and showed no melanin deposition in their oocytes (Fig. 3 .C). TEM revealed that melanosomes formed normally in both wild-type and mitf −/− Xenopus tropicalis oocytes (Fig. 3 .D-G). Thus, the normal melanin deposition in mitf-/- Xenopus tropicalis oocytes indicates that disrupting the bHLH-LZ domain of Mitf does not affect melanogenesis in these oocytes. Conversely, the absence of melanin deposition in tyr −/− Xenopus tropicalis oocytes indicates that melanogenesis in Xenopus tropicalis oocytes depends on Tyr. Both WT and mitf −/− Xenopus tropicalis ovarian membranes exhibited black spots, while tyr −/− Xenopus tropicalis ovarian membranes did not (Fig. 3 .A-C). This suggests that the black spots on the ovarian membranes are likely caused by melanocytes and that melanin synthesis in these spots depends on Tyr. The black spots on the ovarian membranes of WT and tyr −/− Xenopus tropicalis contained numerous melanosomes, which are characteristic organelles of melanocytes, filled with abundant melanin granules (Fig. 3 .H-K). This indicates that the black spots on the ovarian membranes are primarily composed of melanocytes, resulting in their black coloration. Furthermore, this finding implies that the melanocytes forming the black spots on the ovarian membranes may represent a novel type of melanocyte whose development is not regulated by Mitf, warranting further investigation. RNA-seq data indicated that mitf mRNA expression levels were low in both WT and mitf −/− Xenopus tropicalis during oogenesis, with no statistically significant differences observed despite a decreasing trend as oocyte development progressed (Fig. 3 .L). The expression levels of tyr mRNA showed no significant differences between WT and mitf −/− Xenopus tropicalis during oogenesis. In both, tyr mRNA expression significantly decreased as oocyte development proceeded. Specifically, tyr mRNA levels remained stable until stage 3, then markedly decreased at stage 4, continuing to very low levels by stage 6 (Fig. 3 .M). This pattern of tyr mRNA expression is consistent with previous reports on Xenopus laevis oogenesis ( 33 – 36 ). The expression levels of tyrp1 and dct mRNA followed similar patterns to that of tyr mRNA (Supplementary Figure S4 .A and B). These findings suggest that the expression of key melanogenic enzymes peaks at stage 3 during Xenopus tropicalis oogenesis and then gradually decreases, with tyr mRNA expression reaching very low levels after oocyte maturation. Who regulates the expression of tyr mRNA during oocyte melanogenesis? In melanocytes, melanogenesis is primarily controlled by MITF, which regulates key enzymes such as TYR and TYRP1( 14 ). However, during oocyte development in Xenopus tropicalis , mitf mRNA levels are low, and normal melanogenesis occurs in mitf −/− oocytes despite targeting and disrupting the core bHLH-LZ domain (Fig. 3 .A-G) ( 26 ). This suggests that Mitf is not the master regulator of oocyte melanogenesis in Xenopus tropicalis . In vertebrates, the MiT subfamily of transcription factors, including TFEB, TFE3, and TFEC, can form heterodimers with MITF and bind to E-box sequences (typically a 6-bp CANNTG motif) in the promoter regions of target genes like TYR , DCT , TYRP1 , and PMEL ( 14 ). This raises the question: do TFEB, TFEC, or TFE3 directly regulate the expression of key enzymes such as Tyr, Dct, and Tryp1 in mitf −/− Xenopus tropicalis oocytes, or do they compensate for the loss of MITF to ensure normal melanogenesis? During oogenesis in both WT and mitf −/− Xenopus tropicalis , tfeb and tfec mRNA levels were low, whereas tfe3 mRNA levels were relatively high and showed a similar trend to tyr , tyrp1 , and dct mRNA (Fig. 3 .N-O and Supplementary Figure S3 .A-C). There were no significant differences in tfe3 mRNA expression between WT and mitf −/− oocytes (Fig. 3 .N). Additionally, the amino acid sequences of TFE3 and MITF are largely identical (Supplementary Figure S3 .D). We hypothesize that during oocyte development in Xenopus tropicalis , melanin deposition in the animal pole of oocytes may be regulated by one or more members of the MiT subfamily (MITF, TFEB, TFE3, and TFEC). These factors might control the expression of genes such as tyr , dct , and tyrp1 as needed to facilitate melanogenesis (UM2 in Fig. 3 .P). Alternatively, the MiT subfamily may regulate the transcription of these genes into mRNA, which is then stored as maternal RNA in oocytes and degraded or translated as necessary to complete melanogenesis (UM1 in Fig. 3 .P). Further examination of the mRNA expression of melanogenesis-related genes ( oca2 , pmel , slc24a5 , slc45a2 , atp7a , and gpnmb ) revealed patterns similar to those of tfe3 and tyr mRNA (Fig. 4 .A). Specifically, during stages 1 to 3 of oocyte development, these genes maintained relatively high expression levels, which significantly decreased during stages 4 to 6 (Fig. 4 .A). In contrast, the mRNA expression of genes involved in the synthesis of pterinosomes in xanthophores and guanine platelet crystals in iridophores did not exhibit the same pattern as tfe3 and tyr mRNA (Fig. 4 .B-C). This suggests that the synthesis of pigments related to pterinosomes and guanine platelet crystals in Xenopus tropicalis oocytes is likely not regulated by Tfe3. The expression trend of melanogenesis-related genes in Xenopus tropicalis oocytes closely mirrored that of tfe3 mRNA (Fig. 4 .A), indicating a potential regulatory role of Tfe3 in melanogenesis-related gene expression. However, further experimental validation is required. Overall, melanogenesis in Xenopus tropicalis oocytes appears to depend on Tyr rather than Mitf, challenging the previous understanding of Mitf as the master regulator of melanocyte development and melanogenesis and suggesting a different role for mitf in oocyte development. Other significant molecular features during oocyte development in Xenopus tropicalis Transcriptome data from WT and mitf −/− Xenopus tropicalis oocytes were further analyzed to delineate key molecular features during oocyte development. Genes were selected based on a screening criterion of P-value ≤ 0.05 and FPKM ≥ 1, with log2FC ranked in descending order, revealing that the top genes predominantly related to mitochondria (Supplementary Table S3 ). The expression patterns of these mitochondrial-related genes' mRNAs were consistent between WT and mitf −/− Xenopus tropicalis oocytes, showing no significant differences between the two groups (Fig. 5 .A-B and Supplementary Table S3 ). Specifically, expression levels were low during stages 1 and 2, increased significantly by stage 3, peaked at stage 4, and although slightly reduced by stage 6, remained relatively high. During the development of Xenopus tropicalis oocytes, the expression levels of four mitochondrial-related genes, cox1 , cox2 , cox3 , and nd3 , are notably high (Fig. 5 .A-B and Supplementary Table S3 ). Specifically, cox1 , cox2 , and cox3 encode subunits 1, 2, and 3 of cytochrome c oxidase (complex IV), respectively ( 37 ). Subunits 1 and 2 form the catalytic core of complex IV, with subunit 2 transferring electrons from cytochrome c to the active center of subunit 1 ( 37 , 38 ). Subunit 3, though not part of the catalytic core, stabilizes the structure and function of complex IV ( 37 , 38 ). Cytochrome c oxidase, located on the inner mitochondrial membrane, is the terminal enzyme of the mitochondrial electron transport chain. Its primary function is to catalyze the transfer of electrons from cytochrome c to oxygen, producing water and simultaneously pumping protons from the mitochondrial matrix into the intermembrane space ( 37 , 38 ). This creates a transmembrane electrochemical gradient (proton gradient) that drives ATP synthesis. The nd3 gene encodes subunit 3 of NADH dehydrogenase (complex I), the first complex in the mitochondrial electron transport chain ( 39 ). Complex I transfers hydrogen atoms and electrons from NADH to coenzyme Q on the inner mitochondrial membrane ( 39 ). This process also involves the pumping of protons from the mitochondrial matrix into the intermembrane space, contributing to the proton gradient for ATP synthesis ( 39 ). Therefore, during development to stage 3, Xenopus tropicalis oocytes undergo extensive mitochondrial biogenesis and ATP production, indicating a high demand for energy as they progress from stage 3 to mature oocytes. Among the key genes regulating mitochondrial biogenesis and functional homeostasis, mfn1 , mfn2 , and tfam mRNA levels remain relatively high throughout all stages of Xenopus tropicalis oocyte development (Fig. 5 .C-D and Supplementary Table S3 ). These findings indicate that extensive mitochondrial biogenesis is crucial for oocyte development in Xenopus tropicalis . Additionally, the data suggest that the formation of the mitochondrial cloud in these oocytes is tightly regulated by a genetic network( 40 ). Further elucidation of the mechanisms and roles of mitochondrial cloud formation during oocyte development is therefore of significant importance( 40 , 41 ). The formation of the mitochondrial cloud is a significant feature during oocyte development, alongside the notable formation of the zona pellucida( 42 ). The zona pellucida is a transparent, tough, and light-permeable membrane surrounding the oocyte, primarily composed of several glycoproteins forming a clear extracellular matrix( 43 ). This structure encases the oocyte, playing crucial roles in protection, fertilization regulation, and embryo development. The main components of the zona pellucida are glycoproteins, including ZP1, ZP2, ZP3, and ZP4( 42 , 43 ). We examined the expression of homologous genes zp2 , zp3.2 , zp4 , and zp4.2 during Xenopus tropicalis oogenesis. The results showed that although the mRNA levels of zp2 , zp3.2 , and zp4.2 slightly decreased as oocyte development progressed, they remained relatively low without significant changes (Fig. 5 .E-J and Supplementary Table S3 ). In contrast, the expression of zp4 mRNA remained high throughout oogenesis but significantly decreased, particularly from stage 4 to stage 6 (Fig. 5 .E-J). Typically, the mRNAs of zp2 , zp3.2 , zp4 , and zp4.2 are transcribed in follicular cells, translated into corresponding proteins, and subsequently secreted around the oocyte to contribute to the formation of the zona pellucida( 42 – 44 ). However, our transcriptome sequencing detected a substantial presence of zp2 , zp3.2 , zp4 , and zp4.2 transcripts within the oocyte itself (Fig. 5 .E-J), indicating that the oocyte might also express these mRNAs during oogenesis. This finding warrants further experimental validation. Particularly, the changes in zp4 mRNA expression and its relationship with the acrosome reaction merit further investigation( 42 , 45 ). We examined the expression of genes associated with acentriolar spindle assembly during meiosis. The mRNA levels of pcnt and tacc3 increased progressively during oogenesis, with tacc3 mRNA showing relatively high levels (Fig. 5 .K-L and Supplementary Table S3 ). Pericentrin (PCNT) plays a crucial role in oocyte meiosis, being involved in the localization of acentriolar microtubule-organizing centers (aMTOCs), spindle assembly, and chromosome segregation ( 46 ). TACC3 is essential for the localization and function of aMTOCs during meiosis. It promotes microtubule nucleation and stability by interacting with microtubule-associated proteins such as chTOG ( 46 , 47 ). In Xenopus tropicalis , the increasing expression levels of pcnt and tacc3 mRNA during oogenesis suggest that these genes might play a regulatory role in meiosis. Analysis of transcriptome data from WT and mitf −/− Xenopus tropicalis oocytes reveals that the molecular characteristics associated with mitochondrial cloud formation are the most prominent during oocyte development. This finding suggests that the mitochondrial cloud is crucial for oocyte maturation. Similar subcellular structures have been observed in the oocytes of various vertebrates, including humans, mice, and zebrafish, indicating evolutionary conservation( 40 , 41 ). Thus, the Xenopus tropicalis mitochondrial cloud serves as an excellent model for studying its role and mechanisms in oocyte development. Furthermore, the expression patterns of zona pellucida proteins ( zp2 , zp3.2 , zp4 , and zp4.2 ) and meiosis-related genes ( pcnt and tacc3 ) underscore the need to investigate their roles in oocyte development. Therefore, using Xenopus tropicalis as a model organism to study oogenesis is crucial for understanding reproductive biology. Discussions The deposition of animal pole pigment in Xenopus tropicalis oocytes is crucial for their development, influencing oocyte polarity, growth, and subsequent embryonic development. In this study, we disrupted the bHLH-LZ domain of Mitf in Xenopus tropicalis , resulting in mitf −/− frogs that lack melanocytes and show significantly reduced expression of key melanogenesis genes, indicating a loss of Mitf activity. However, mitf −/− oocytes still regulate tyr mRNA expression to complete melanogenesis, leading to melanin deposition at the animal pole. In contrast, tyr −/− oocytes show no melanin deposition at the animal pole. Thus, melanogenesis in Xenopus tropicalis oocytes depends on Tyr rather than Mitf, potentially due to other MiT subfamily factors regulating the expression of tyr , dct , and tyrp1 during oogenesis. We also found that mitochondrial cloud formation represents the most significant molecular change during oocyte development, alongside the expression patterns of zona pellucida proteins and meiosis-related genes such as pcnt and tacc3 . These findings suggest that further elucidation of the Tyr-dependent, Mitf-independent mechanisms of animal pole pigment deposition will enhance our understanding of melanogenesis and oocyte development. Additionally, Xenopus tropicalis oocytes will serve as an effective model for studying the mitochondrial cloud, zona pellucida, meiosis, oogenesis, and reproductive medicine. The MiT family, a group of bHLH-Zip transcription factors, primarily includes MITF, TFEB, TFEC, and TFE3( 14 , 48 ). These transcription factors typically bind to E-box sequences (CANNTG) via their bHLH-Zip domain to regulate target gene transcription. They can form homodimers (e.g., MITF-MITF) or heterodimers (e.g., MITF-TFEB), enhancing their DNA-binding ability and specificity( 14 , 48 ). Consequently, they play crucial roles in gene expression, cell differentiation, metabolism, and autophagy. Tfec plays a direct role in regulating zebrafish iridophore development starting from the multipotent pigment cell progenitor stage( 49 ). It is also essential for iridophore development in a lizard model( 50 ). Therefore, it is hypothesized that Tfec regulates melanocyte development by influencing the differentiation of multipotent pigment cell progenitors, thereby affecting melanogenesis. Although TFEB shares conserved domains with MITF, there are few reports of TFEB directly regulating melanogenesis( 51 , 52 ). However, TFEB may influence melanogenesis through autophagy and lysosomal biogenesis( 52 ). Research indicates that MITF induction of UVRAG is crucial for UV-induced tanning, while inactivation of TFEB or TFE3 has minimal effect on UVRAG expression following α-MSH stimulation( 53 ). This implies that TFEB and TFE3 likely do not regulate melanogenesis via the α-MSH pathway, but confirms UVRAG's role in melanogenesis regulation. UVRAG, a multifunctional protein, participates in autophagy, endocytosis, and DNA damage repair( 54 ). In melanogenesis, UVRAG maintains the localization and stability of BLOC-1 to facilitate melanogenic cargo sorting and delivery( 53 ). TFEB, a pivotal transcription factor in autophagy and lysosomal biogenesis, activates autophagy-related genes by binding to E-box sequences, crucial for autophagosome formation, elongation, and maturation( 52 , 55 ). UVRAG interacts with the class C Vps complex, a key component of the endosomal fusion machinery, forming the UVRAG-class-C-Vps complex. This interaction stimulates RAB7 GTPase activity and promotes the fusion of autophagosomes with late endosomes/lysosomes, enhancing the delivery and degradation of autophagic cargo and accelerating endosome–endosome fusion, leading to rapid degradation of endocytic cargo( 54 ). Therefore, TFEB may impact melanogenesis by modulating the interaction between UVRAG and BLOC-1, which is influenced by the stability of the UVRAG-class-C-Vps complex regulated by autophagosomes( 53 , 54 ). Although TFE3 can’t regulate melanogenesis via the α-MSH signaling pathway( 53 ), Tfe3a can restore melanophore development at a very low frequency in zebrafish( 56 ). Additionally, TFE3 interacts with p300, further participating in melanogenesis-related signaling pathways( 57 ). Studies have shown that Tfe3 can activate the expression of Tyr and Tyrp1( 58 ). Therefore, overall, although Tfeb, Tfec, and Tfe3 may be involved in the regulation of oocyte melanogenesis, considering that Tfe3 has a higher expression level in Xenopus tropicalis oocytes compared to the very low levels of Tfeb and Tfec, and considering the evidence that Tfe3 regulates Tyr and Tyrp1 expression, we hypothesize that Tfe3, rather than Mitf, regulates melanogenesis-related gene expression during oogenesis in Xenopus tropicalis . This regulation results in melanin deposition at the animal pole of the oocyte. Further experimental data elucidating the molecular mechanisms of melanin deposition in Xenopus tropicalis oocytes will enhance research in oogenesis and reproductive medicine. Methods and Materials Xenopus tropicalis maintenance and husbandry Adult Xenopus tropicalis frogs were obtained from Nasco (Fort Atkinson, WI, USA; http://www.enasco.com ). The maintenance and husbandry of the frogs, as well as the procurement of their oocytes, adhered to established methods( 26 , 59 ). All experiments involving Xenopus tropicalis were approved by the Institutional Animal Care and Use Committee of the Southern University of Science and Technology. The procurement of Xenopus tropicalis oocytes The establishment of mitf −/− and tyr −/− Xenopus tropicalis lines has been reported in our previous studies( 26 ). The method for collecting oocytes from WT, mitf −/− , and tyr −/− Xenopus tropicalis was slightly modified from the protocol described in the literature( 60 ). First, healthy adult WT, mitf −/− , and tyr −/− Xenopus tropicalis were selected. To reduce stress, the frogs were anesthetized with 0.2% MS-222 before the procedure. The abdomen was disinfected with 70% ethanol, and a small incision (approximately 1–2 cm) was made in the center of the abdomen, taking care not to cut too deeply to avoid damaging internal organs. The ovaries were carefully removed through the incision, and a portion of the ovarian tissue was gently excised using scissors or forceps. The excised ovarian tissue was placed in a dish containing cold 1× MBS solution to prevent damage to the oocytes. The ovarian tissue was then repeatedly washed in 1× MBS solution to remove blood and debris. After washing, the ovarian tissue was transferred to 1× MBS solution containing 1.5 mg/mL collagenase and incubated at 25°C for 1–5 hours with gentle agitation to facilitate the release of oocytes from the ovarian tissue. The process was monitored under a stereomicroscope to ensure that the follicle cells were completely separated from the oocytes. Following incubation, the oocytes were washed multiple times with fresh 1× MBS solution to remove residual collagenase and debris. The oocytes were then observed under a stereomicroscope, and oocytes at various developmental stages were selected. RNA-seq library preparation and RNA-seq analysis Oocytes were collected from WT and mitf −/− adult frogs, with each group comprising two replicates. Oocytes were disrupted using a disposable syringe until completely lysed, and total RNA was extracted following the manufacturer’s instructions for the TransZol Up lysis reagent (ET111-01, TransGen Biotech). The extracted RNA samples were submitted to Novogene for RNA-seq library preparation and sequencing. RNA-seq analysis was conducted by Wuhan Frasergen Information Co., Ltd. The previously published protocol guided the RNA-seq data analysis( 61 ). Briefly, SOAPnuke software (v2.1.0) was used to filter raw reads to obtain clean reads. Clean reads were aligned to the Xenopus tropicalis reference genome UCB_Xtro_10.0 using HISAT2. Bowtie2 software then mapped the quality-controlled sequences to the reference transcriptome. RSEM analyzed the Bowtie2 alignment results to determine the number of reads mapped to each transcript and calculate FPKM (Fragments Per Kilobase per Million bases) values. Differential expression analysis was performed using DESeq2. RNA-seq data has been publicly deposited to Genome Sequence Archive ( https://ngdc.cncb.ac.cn/gsa/ ) with the accession number CRA017994 (Reviewers can obtain the RNA-seq data through this link: https://ngdc.cncb.ac.cn/gsa/s/1WKfk9MH ). Hematoxylin-Eosin staining, transmission electron microscopy (TEM) The samples underwent histological analysis, starting with fixation in FAS eyeball fixative (Servicebio, Wuhan, China) at room temperature for 24 hours. The fixed samples were then dehydrated using graded ethanol (75%, 85%, 95%, and 100%), followed by replacement with xylene and embedding in paraffin wax. Sections with a thickness of 6 µm were prepared from the embedded tissues and subsequently dewaxed in xylene. Rehydration was carried out using graded ethanol concentrations (100%, 95%, and 70%) before staining with the hematoxylin-eosin staining kit (Baso, Zhuhai, China) and immunofluorescent staining. Hematoxylin-eosin-stained results were captured using an Olympus BX53 upright microscope (Olympus, Japan). Our previously published protocol guided the TEM sample preparation( 26 ). Briefly, Xenopus tropicalis tissue samples, sized 1 mm × 1 mm, were fixed overnight at 4°C in 2% glutaraldehyde. Oocytes were also fixed overnight at 4°C in 2% glutaraldehyde. Following fixation, the samples underwent four 10-minute washes with 10 mM PBS solution. Subsequently, the samples were fixed with 1% osmium tetroxide at room temperature for 3 hours. The samples were then washed twice with 10 mM PBS solution for 10 minutes each, followed by two washes with ddH 2 O for 10 minutes each. After rinsing with ddH 2 O, the samples were treated with a 2% uranyl acetate solution at room temperature for 2 hours or overnight at 4°C. Following this, the samples were washed four or more times with ddH 2 O for 10 minutes each until the rinsed water was clear. Post-rinsing, the samples were dehydrated using a graded acetone series: 30% acetone once, 50% acetone once, 75% acetone once, and 100% acetone twice, each for 10 minutes. Simultaneously with the gradient dehydration, epoxy resin (9.8 g), DDSA (5.6 g), NMH (4.6 g), and DMP-30 (0.28 mL) were thoroughly mixed at room temperature using a rotary mixer for at least 4 hours to prepare a 100% resin mixture. Then, at room temperature, the samples were treated with 25% resin-acetone once, 50% resin-acetone once, 75% resin-acetone once, and 100% resin once, each for 1.5-2 hours. Following these steps, new 100% resin was added, and the samples were left overnight at 4°C. Unused 100% resin was sealed with parafilm and stored at 4°C for later use. The next day, the samples were embedded in new 100% resin. Subsequently, the embedded samples were baked in a 60°C oven for 2 days to complete the preparation of TEM samples for ultrathin sectioning. Finally, the prepared TEM samples were sent to Wuhan Servicebio Technology Co., Ltd. for ultrathin sectioning, TEM observation, and photography. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All study data are included in the article and/or supplemental information. No other scripts and software were used other than those mentioned in the Methods section. Competing interests The authors declare no competing interests. Funding This work was supported by the National Natural Science Foundation of China (Nos. 32300659, 92169119, 82070420), Shenzhen Science and Technology Innovation Commission Project (No. JCYJ20230807143302004), Shanghai Science and Technology Innovation Action Plan (No. 22N31900800), Shenzhen Clinical Research Center for Emerging Infectious Diseases (No. LCYSSQ20220823091203007), Shenzhen High-level Hospital Construction Fund (No. XKJS-CRGRK-008), and National Key Research and Development Program of China (Nos. 2022YFC2304401，2022YFC2304402). Authors' contributions R.R. and H.L. and Y.C. and W.L. conceived the project. W.L. and J.H. and J.S. performed the experiments and analyzed the data together with S.Y. and H.L. and J.D. and X.F.. S.H. and W.C. contributed TEM data collation and analysis. R.R. and H.Y. wrote the manuscript with input from all the authors. All authors read and approved the final manuscript. 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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. 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 S4. 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/. SupplementaryTableS1.xlsx Supplementary Tables Supplementary Table S1. Gene expression profiles identified through bulk RNA-seq in the dorsal and ventral skin samples of WT (Mcon) and mitf -/- (MKO) Xenopus tropicalis . SupplementaryTableS2.xlsx Supplementary Table S2. Gene expression profiles identified through bulk RNA-seq in the eye samples of wild-type WT (Mcon) and mitf -/- (MKO) Xenopus tropicalis . SupplementaryTableS3.xlsx Supplementary Table S3. Gene expression profiles identified through bulk RNA-seq in the oocytes samples of wild-type WT (Mcon) and mitf -/- (MKO) Xenopus tropicalis . Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Sep, 2024 Reviews received at journal 23 Sep, 2024 Reviews received at journal 13 Sep, 2024 Reviews received at journal 13 Sep, 2024 Reviewers agreed at journal 07 Sep, 2024 Reviewers agreed at journal 06 Sep, 2024 Reviewers agreed at journal 05 Sep, 2024 Reviewers invited by journal 01 Aug, 2024 Editor assigned by journal 30 Jul, 2024 Submission checks completed at journal 29 Jul, 2024 First submitted to journal 26 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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17:10:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":853211,"visible":true,"origin":"","legend":"\u003cp\u003eThe 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.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/319931eee033080e77a7c9bb.png"},{"id":63064217,"identity":"f3ec5c9b-237e-4c06-8d65-154697d5e189","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170156,"visible":true,"origin":"","legend":"\u003cp\u003eXenopus 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 and 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.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/0bed43e5c32cd8229e737d83.png"},{"id":63064219,"identity":"c2b33ef5-e3bd-4431-864a-f1d5c4e71260","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":698981,"visible":true,"origin":"","legend":"\u003cp\u003eTyr regulates melanin deposition in Xenopus tropicalis oocytes. A-C, representative images show the ovarian from one-year-old WT, mitf-/-, and tyr-/- female Xenopus tropicalis, with three frogs observed for each genotype. In Panels 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 and B and C are 0.5 mm, and in the lower panels, they are 0.1 mm. D and 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 Panels D and F, respectively. H and J, figures display TEM images of black spots on the ovarian peritoneum of one-year-old WT and mitf-/- Xenopus tropicalis. Panels I and K are enlarged views of the regions indicated by red arrows in Panels H and J, respectively. The scale bars are as shown in the images. All images in Panels 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 Figures L-O, please refer to Supplementary Table S3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/c2c694b96404ebe314eb6152.png"},{"id":63064681,"identity":"aa05bee2-857d-4daf-8724-78554640a621","added_by":"auto","created_at":"2024-08-22 17:18:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175697,"visible":true,"origin":"","legend":"\u003cp\u003eThe 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 and 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 Supplementary Table S3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/dfe7563771f17948443f7102.png"},{"id":63064221,"identity":"656ba1aa-0446-4086-9fca-68c5050bf2c7","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108822,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially 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.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/4808e8ff96069eb6e276ceb7.png"},{"id":63065180,"identity":"186ef31a-7bd5-4999-87f0-40999e0c1546","added_by":"auto","created_at":"2024-08-22 17:26:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2993482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/51a18941-b0dd-4b10-b9c0-5434cdf8c0d3.pdf"},{"id":63064223,"identity":"20c8e785-57b2-4240-b196-2082f99b284a","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1233448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1\u003c/strong\u003e. Transcripts of human\u0026nbsp;\u003cem\u003eMITF\u003c/em\u003e\u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) and mouse\u0026nbsp;\u003cem\u003eMitf\u003c/em\u003e\u0026nbsp;(\u003cstrong\u003eB\u003c/strong\u003e). For detailed information, refer to NCBI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S2\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;In\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emitf\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e\u0026nbsp;Xenopus tropicalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, the bHLHzip domain of the Mitf protein, is inactivated\u003c/strong\u003e.\u0026nbsp;\u003cstrong\u003eA,\u0026nbsp;\u003c/strong\u003ethe\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003epicture provides a schematic representation of the transcripts and related information of the\u0026nbsp;\u003cem\u003emitfa\u003c/em\u003e\u0026nbsp;gene locus in zebrafish and the\u0026nbsp;\u003cem\u003emitf\u003c/em\u003e\u0026nbsp;gene locus in\u0026nbsp;\u003cem\u003eXenopus tropicalis\u003c/em\u003e. The red arrow indicates the penultimate exon, which is responsible for transcribing a common part of the transcripts from this gene locus.\u0026nbsp;\u003cstrong\u003eB\u003c/strong\u003e\u0026nbsp;and\u0026nbsp;\u003cstrong\u003eC\u003c/strong\u003e, 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\u0026nbsp;\u003cem\u003eXenopus tropicalis\u0026nbsp;\u003c/em\u003eMitf protein and the human MITF protein. The red arrow points to the knockout site in the\u0026nbsp;\u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u0026nbsp;Xenopus tropicalis\u003c/em\u003e\u0026nbsp;Mitf protein. 'N' indicates the amino-terminal, and 'C' indicates the carboxy-terminal.\u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e\u0026nbsp;and\u0026nbsp;\u003cstrong\u003eE\u003c/strong\u003e, 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\u0026nbsp;\u003cstrong\u003eD\u0026nbsp;\u003c/strong\u003eand\u0026nbsp;\u003cstrong\u003eE\u003c/strong\u003e, 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\u0026nbsp;\u003cstrong\u003eA\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e, and\u0026nbsp;\u003cstrong\u003eE\u003c/strong\u003e\u0026nbsp;indicate the knockout site of\u0026nbsp;\u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u0026nbsp;Xenopus tropicalis\u003c/em\u003e\u0026nbsp;\u003cem\u003emitf\u003c/em\u003e. For detailed data analysis, refer to NCBI (https://www.ncbi.nlm.nih.gov/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S3\u003c/strong\u003e.\u0026nbsp;\u003cstrong\u003eDifferential expression of genes related to RPE cells and melanocytes in the eyes of WT and\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emitf\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eXenopus tropicalis\u003c/strong\u003e\u003c/em\u003e. Mcon denotes WT\u0026nbsp;\u003cem\u003eXenopus tropicalis\u003c/em\u003e\u0026nbsp;eye samples, while MKO denotes\u0026nbsp;\u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u0026nbsp;Xenopus tropicalis\u003c/em\u003e\u0026nbsp;eye samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S4\u003c/strong\u003e.\u0026nbsp;\u003cstrong\u003eA-C\u003c/strong\u003e, Expression levels of\u0026nbsp;\u003cem\u003etfeb\u003c/em\u003e,\u0026nbsp;\u003cem\u003etyrp1\u003c/em\u003e, and\u0026nbsp;\u003cem\u003edct\u003c/em\u003e\u0026nbsp;mRNA during oocyte development in WT and\u003cem\u003e\u0026nbsp;mitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u0026nbsp;Xenopus tropicalis\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e, Comparison of amino acid sequences of human MITF-A,\u0026nbsp;\u003cem\u003eXenopus tropicalis\u003c/em\u003e\u0026nbsp;Mitf, and\u0026nbsp;\u003cem\u003eXenopus tropicalis\u003c/em\u003e\u0026nbsp;Tfe3 proteins. Protein sequence information was obtained from https://www.uniprot.org/.\u003c/p\u003e","description":"","filename":"SupplementaryFiguresandFigurelegends.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/dc1971528989f8be85c93cf6.pdf"},{"id":63064225,"identity":"1c05c325-5db3-4469-b60d-1ff8140f2b8c","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11040179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Tables\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S1. \u003c/strong\u003eGene expression profiles identified through bulk RNA-seq in the dorsal and ventral skin samples of WT (Mcon) and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (MKO) \u003cem\u003eXenopus tropicalis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/65a8f49aebc799f3fc7bbb6f.xlsx"},{"id":63064226,"identity":"47cff3f8-fdba-4ce5-873d-e7fc7d82fa83","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7214404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S2. \u003c/strong\u003eGene expression profiles identified through bulk RNA-seq in the eye samples of wild-type WT (Mcon) and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (MKO) \u003cem\u003eXenopus tropicalis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/e211f89dcfce31834cb1040a.xlsx"},{"id":63064224,"identity":"a5845bd2-cc05-4f63-8d3e-9d51d6f52185","added_by":"auto","created_at":"2024-08-22 17:10:02","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":7240146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S3. \u003c/strong\u003eGene expression profiles identified through bulk RNA-seq in the oocytes samples of wild-type WT (Mcon) and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (MKO) \u003cem\u003eXenopus tropicalis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4807093/v1/12c8e75ebf769be47e33c7d3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dispensable role of mitf in melanogenesis of Xenopus tropicalis oocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOogenesis, the process by which oocytes (egg cells) develop, involves complex interactions among genetic, biochemical, and environmental factors(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In some species, oocytes undergo melanin deposition during this process. Melanin pigmentation in oocytes is a critical feature that affects not only the aesthetic attributes of the eggs but also their survival and fitness in natural environments(\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). For instance, melanin pigmentation can protect the developing oocyte from UV radiation and oxidative stress, thereby contributing to the overall viability of the embryo(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, melanin pigment granules may play a role in the thermal regulation of the eggs, influencing embryonic development rates(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Therefore, understanding the molecular mechanisms governing melanin synthesis in oocytes provides insights into oocyte development and other broader biological phenomena, such as gene regulatory networks, organelle biogenesis, cellular differentiation, and intracellular transport.\u003c/p\u003e \u003cp\u003eIn vertebrates, melanogenesis primarily occurs in melanocytes, the retinal pigment epithelium (RPE), and the oocytes of certain species(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The melanogenesis process involves a series of enzymatic reactions that begin with the amino acid tyrosine(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Central to melanogenesis is the enzyme tyrosinase, which catalyzes the initial and rate-limiting steps of melanin synthesis, converting tyrosine into dihydroxyphenylalanine (DOPA) and subsequently into dopaquinone(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Further complex biochemical transformations lead to the production of different types of melanin, mainly eumelanin (brown to black pigment) and pheomelanin (yellow to red pigment)(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The type and amount of melanin produced are regulated by genetic, hormonal, and environmental factors, making melanogenesis a highly dynamic and tightly controlled process.\u003c/p\u003e \u003cp\u003eThe molecular regulation of melanogenesis is predominantly controlled by the microphthalmia-associated transcription factor (MITF), a master regulator in melanocytes that governs the expression of essential melanogenic enzymes and structural proteins(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). MITF specifically regulates enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2)(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). MITF activity is modulated by several signaling pathways, including cAMP/protein kinase A (PKA), Wnt/β-catenin, and mitogen-activated protein kinase (MAPK)(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These pathways are activated by various extracellular signals, such as UV radiation, hormones, and cytokines, which collectively modulate MITF expression and activity, highlighting the complexity of melanin production regulation. Despite significant advances in understanding the molecular mechanisms of melanogenesis(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), many aspects remain unclear. The roles of non-coding RNAs, epigenetic modifications, and genetic polymorphisms in melanogenic pathways are areas of ongoing research(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is noteworthy that although melanogenesis plays a crucial role in \u003cem\u003eXenopus\u003c/em\u003e oocyte development, the molecular regulatory mechanisms of \u003cem\u003eXenopus\u003c/em\u003e oocyte melanogenesis remain largely unknown(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Previous research has shown that tyrosinase activity is markedly higher in stage III and stage IV \u003cem\u003eXenopus\u003c/em\u003e oocytes compared to other stages(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Additionally, early studies have found that tyrosinase activity is present in both albino and wild-type \u003cem\u003eXenopus\u003c/em\u003e oocytes, with albino oocytes exhibiting higher enzyme activity(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Numerous experiments have confirmed that the tyrosinase inhibitor PTU can induce the production of melanin-free oocytes and tadpoles in \u003cem\u003eXenopus\u003c/em\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Furthermore, gene-editing technologies such as TALEN and CRISPR/Cas9 have produced tyrosinase knockout lines of \u003cem\u003eXenopus laevis\u003c/em\u003e and \u003cem\u003eXenopus tropicalis\u003c/em\u003e(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, the molecular mechanisms regulating tyrosinase expression in oocytes remain unclear. Due to the significant differences between the intracellular and extracellular environments of oocytes and melanocytes(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), the similarities and differences in the molecular regulatory mechanisms of melanogenesis in oocytes and melanocytes are still unknown.\u003c/p\u003e \u003cp\u003eIn our previous research, we successfully established \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e lines using CRISPR/Cas9 gene editing technology(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). We discovered that while \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes completely lacked melanin deposition, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes exhibited normal melanin deposition(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This unexpected oocyte phenotype further motivated us to study the molecular regulatory mechanisms of oocyte melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e. Therefore, in this study, we utilized oocytes from three genotypes of \u003cem\u003eXenopus tropicalis\u003c/em\u003e: WT, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, to investigate the molecular characteristics of oocyte melanogenesis. Our results revealed that although the core enzymes involved in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocyte melanogenesis are tyrosinase, tyrosinase-related protein 1 (Trp1), and tyrosinase-related protein 2 (Trp2), the master regulator of these core enzymes' expression is not Mitf. Thus, our findings suggest that \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocyte melanogenesis depends on a master regulator other than Mitf, revising our understanding of the function of the \u003cem\u003emitf\u003c/em\u003e gene and highlighting the importance of further exploring the molecular regulatory mechanisms of oocyte melanogenesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDisruption of the Mitf basic helix-loop-helix leucine zipper domain impairs its function\u003c/h2\u003e \u003cp\u003eMicrophthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family, is highly conserved and serves as a pivotal regulator in numerous biological processes, including cellular differentiation, proliferation, and survival across various tissues(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Initially identified for its role in ocular development, MITF has since emerged as a multifaceted transcription factor with implications extending far beyond its namesake. Its intricate involvement in melanocyte biology, osteoclastogenesis, and immune response underscores its versatile functionality(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Structurally, MITF exhibits a modular organization characterized by distinct domains responsible for DNA binding (the bHLH-LZ domain binds DNA as dimers), protein-protein interactions, and transcriptional activation (a strong transcription activation domain at the N-terminus and a much weaker second transactivation domain at the C-terminus) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These domains intricately cooperate to confer specificity in target gene recognition, recruitment of cofactors, and modulation of transcriptional activity(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). When both transcription activation domains (TADs) of MITF are inactivated, the mutated MITF can form dimers with wild-type MITF, exerting a dominant negative effect(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Given that the bHLH-LZ domain is primarily responsible for the DNA-binding function of MITF, disrupting this domain results in the loss of MITF function(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Therefore, we utilized CRISPR/Cas9 gene editing technology to disrupt the bHLH-LZ domain of Mitf in \u003cem\u003eXenopus tropicalis\u003c/em\u003e to achieve \u003cem\u003emitf\u003c/em\u003e gene knockout(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, the transcription of the \u003cem\u003eMITF\u003c/em\u003e gene locus mRNA in mammals is complex and variable (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, NCBI records show only one \u003cem\u003emitf\u003c/em\u003e transcript for \u003cem\u003eXenopus tropicalis\u003c/em\u003e and three \u003cem\u003emitf\u003c/em\u003e transcripts for zebrafish (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.A). This discrepancy may be due to limited research on \u003cem\u003emitf\u003c/em\u003e mRNA transcription in these species. Analysis of \u003cem\u003eMITF\u003c/em\u003e mRNA transcription in humans, mice, \u003cem\u003eXenopus tropicalis\u003c/em\u003e, and zebrafish reveals variability at the 5' end and consistency at the 3' end (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.A). Within the same species, the final few exons encoding these isoforms are identical, resulting in all MITF protein isoforms having the same C-terminus, likely due to the presence of highly conserved bHLH-LZ and TAD domains in these regions(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). To disrupt all isoforms encoded by the \u003cem\u003eXenopus tropicalis mitf\u003c/em\u003e gene locus, we designed a Cas9-targeted knockout site on the penultimate exon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.A-C). According to Alphafold predictions, the bHLH-LZ and TAD domains are highly conserved between \u003cem\u003eXenopus tropicalis\u003c/em\u003e Mitf and human MITF (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.B-C). The DNA and polypeptide binding sites within the bHLH-LZ domain exhibit significant conservation across species, and our designed guide RNA targets these conserved sites (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.D-E). This suggests that in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, the core bHLH-LZ domain of \u003cem\u003eMitf\u003c/em\u003e isoforms is disrupted, impairing their function. Indeed, following CRISPR/Cas9 knockout of \u003cem\u003emitf\u003c/em\u003e in \u003cem\u003eXenopus tropicalis\u003c/em\u003e, the resulting \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e frogs lack melanocytes, xanthophores, and granular glands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B-F)(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). During vertebrate eye development, various Mitf isoforms play crucial regulatory roles. Studies on various Mitf mutant mouse strains demonstrate that mutations in the bHLH-LZ domain of mouse MITF lead to abnormal eye development, including smaller eyes and aberrant pigmentation(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In contrast, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e exhibit normal eye size with apparently normal eye pigmentation(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Given the complexity of MITF's regulatory role in eye development and the subtle nature of some phenotypic abnormalities(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), further research is needed to assess whether the retina and RPE development in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e are truly unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F). Overall, these data suggest that in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, the disruption of the core bHLH-LZ domain of Mitf impairs the critical regulatory functions of various Mitf isoforms, particularly in melanocyte development and melanogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAbsence of melanogenesis in the skin of\u003c/b\u003e \u003cb\u003emitf\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eXenopus tropicalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the molecular changes in Mitf-regulated target gene expression in \u003cem\u003emitf\u003c/em\u003e knockout \u003cem\u003eXenopus tropicalis\u003c/em\u003e, we performed Bulk RNA-seq on dorsal and ventral skin samples from adult wild-type (WT) and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e frogs. It is well-established that genes involved in melanogenic enzymes (e.g., \u003cem\u003eTYR\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, \u003cem\u003eDCT\u003c/em\u003e), melanosome ionic equilibrium (e.g., \u003cem\u003eSLC45A2\u003c/em\u003e, \u003cem\u003eSLC24A5\u003c/em\u003e), melanocyte development signaling pathways (e.g., \u003cem\u003eMC1R\u003c/em\u003e), and melanosome biogenesis (e.g., \u003cem\u003eMLANA\u003c/em\u003e, \u003cem\u003ePMEL\u003c/em\u003e) are regulated by MITF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A). We first assessed the expression of these melanogenesis-related target genes. As expected, the expression levels of \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003etyrp1\u003c/em\u003e, \u003cem\u003edct\u003c/em\u003e, \u003cem\u003eslc45a2\u003c/em\u003e, \u003cem\u003eslc24a5\u003c/em\u003e, \u003cem\u003emc1r\u003c/em\u003e, \u003cem\u003emlana\u003c/em\u003e, and \u003cem\u003epmel\u003c/em\u003e were significantly reduced in both dorsal and ventral skin samples of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B-C and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additionally, melanocyte marker genes such as \u003cem\u003eslc24a4\u003c/em\u003e, \u003cem\u003eednrb2\u003c/em\u003e, \u003cem\u003ekcnj13\u003c/em\u003e, \u003cem\u003etrpm1\u003c/em\u003e, and \u003cem\u003emlph\u003c/em\u003e also exhibited significantly reduced expression in the skin samples of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e frogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B-C and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We then examined the expression of these genes in eye samples from adult WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e. However, the expression of melanogenesis-related genes was not consistently reduced in the eyes of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e frogs (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This discrepancy may be attributed to the presence of RPE cells in the retina, despite the absence of melanocytes in the choroid of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In mammals, MITF significantly influences the development of the retinal pigment epithelium (RPE) and retina by regulating the expression of genes such as \u003cem\u003eRDH5\u003c/em\u003e, \u003cem\u003eRLBP1\u003c/em\u003e, \u003cem\u003eMSI2\u003c/em\u003e, \u003cem\u003eDAPL1\u003c/em\u003e, miR204/211, \u003cem\u003eNF2\u003c/em\u003e, \u003cem\u003eBEST1\u003c/em\u003e, \u003cem\u003ePGC1α\u003c/em\u003e, \u003cem\u003ePEDF\u003c/em\u003e (\u003cem\u003eSERPINF1\u003c/em\u003e), \u003cem\u003ePMEL\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, and \u003cem\u003eTYR\u003c/em\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes, the expression levels of \u003cem\u003erdh5\u003c/em\u003e, \u003cem\u003erlbp1\u003c/em\u003e, \u003cem\u003emsi2\u003c/em\u003e, \u003cem\u003edapl1\u003c/em\u003e, miR204/211, \u003cem\u003enf2\u003c/em\u003e, \u003cem\u003ebest1\u003c/em\u003e, \u003cem\u003epgc1α\u003c/em\u003e, and \u003cem\u003epedf\u003c/em\u003e did not show significant changes compared to wild-type eyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.D-K and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Similarly, the expression of melanogenesis-related genes such as \u003cem\u003epmel\u003c/em\u003e, \u003cem\u003etyrp1\u003c/em\u003e, and \u003cem\u003etyr\u003c/em\u003e, which are regulated by MITF, were not significantly decreased in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Additionally, there was no significant difference in the expression of \u003cem\u003erpe65\u003c/em\u003e, a gene crucial for RPE cells, between wild-type and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These findings indicate that RPE cells in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes can still develop into functional RPE cell layers despite the disruption of the Mitf bHLH-LZ domain(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, the absence of melanocytes in the choroid layer of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e eyes confirms the lack of melanocyte development and melanogenesis(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In conclusion, this molecular evidence demonstrates that disrupting the Mitf bHLH-LZ domain in \u003cem\u003eXenopus tropicalis\u003c/em\u003e leads to a loss of Mitf activity, resulting in the absence of melanocytes and melanogenesis in the skin samples of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, thereby contributing to their colorless and transparent appearance(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNormal oocyte melanin deposition in\u003c/b\u003e \u003cb\u003emitf\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eXenopus tropicalis\u003c/b\u003e \u003cb\u003eduring oogenesis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, the absence of melanocyte development in the skin led us to predict that oocytes in female \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e would also lack melanin deposition(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, contrary to our prediction, melanin deposition in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes was normal, with their pigmentation pattern being almost indistinguishable from that of wild-type oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-B) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Additionally, the ovarian membrane of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, which encases the ovaries, exhibited black spots similar to those observed in wild-type ovarian membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-B). In contrast, \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e lacked black spots on the ovarian membrane and showed no melanin deposition in their oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C). TEM revealed that melanosomes formed normally in both wild-type and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.D-G). Thus, the normal melanin deposition in mitf-/- Xenopus tropicalis oocytes indicates that disrupting the bHLH-LZ domain of Mitf does not affect melanogenesis in these oocytes. Conversely, the absence of melanin deposition in \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes indicates that melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes depends on Tyr. Both WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e ovarian membranes exhibited black spots, while \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e ovarian membranes did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-C). This suggests that the black spots on the ovarian membranes are likely caused by melanocytes and that melanin synthesis in these spots depends on Tyr. The black spots on the ovarian membranes of WT and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e contained numerous melanosomes, which are characteristic organelles of melanocytes, filled with abundant melanin granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.H-K). This indicates that the black spots on the ovarian membranes are primarily composed of melanocytes, resulting in their black coloration. Furthermore, this finding implies that the melanocytes forming the black spots on the ovarian membranes may represent a novel type of melanocyte whose development is not regulated by Mitf, warranting further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRNA-seq data indicated that \u003cem\u003emitf\u003c/em\u003e mRNA expression levels were low in both WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e during oogenesis, with no statistically significant differences observed despite a decreasing trend as oocyte development progressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.L). The expression levels of \u003cem\u003etyr\u003c/em\u003e mRNA showed no significant differences between WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e during oogenesis. In both, \u003cem\u003etyr\u003c/em\u003e mRNA expression significantly decreased as oocyte development proceeded. Specifically, \u003cem\u003etyr\u003c/em\u003e mRNA levels remained stable until stage 3, then markedly decreased at stage 4, continuing to very low levels by stage 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.M). This pattern of \u003cem\u003etyr\u003c/em\u003e mRNA expression is consistent with previous reports on \u003cem\u003eXenopus laevis\u003c/em\u003e oogenesis (\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The expression levels of \u003cem\u003etyrp1\u003c/em\u003e and \u003cem\u003edct\u003c/em\u003e mRNA followed similar patterns to that of \u003cem\u003etyr\u003c/em\u003e mRNA (Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.A and B). These findings suggest that the expression of key melanogenic enzymes peaks at stage 3 during \u003cem\u003eXenopus tropicalis\u003c/em\u003e oogenesis and then gradually decreases, with \u003cem\u003etyr\u003c/em\u003e mRNA expression reaching very low levels after oocyte maturation.\u003c/p\u003e \u003cp\u003eWho regulates the expression of \u003cem\u003etyr\u003c/em\u003e mRNA during oocyte melanogenesis? In melanocytes, melanogenesis is primarily controlled by MITF, which regulates key enzymes such as TYR and TYRP1(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, during oocyte development in \u003cem\u003eXenopus tropicalis\u003c/em\u003e, \u003cem\u003emitf\u003c/em\u003e mRNA levels are low, and normal melanogenesis occurs in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e oocytes despite targeting and disrupting the core bHLH-LZ domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A-G) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This suggests that Mitf is not the master regulator of oocyte melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e. In vertebrates, the MiT subfamily of transcription factors, including TFEB, TFE3, and TFEC, can form heterodimers with MITF and bind to E-box sequences (typically a 6-bp CANNTG motif) in the promoter regions of target genes like \u003cem\u003eTYR\u003c/em\u003e, \u003cem\u003eDCT\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, and \u003cem\u003ePMEL\u003c/em\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This raises the question: do TFEB, TFEC, or TFE3 directly regulate the expression of key enzymes such as Tyr, Dct, and Tryp1 in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes, or do they compensate for the loss of MITF to ensure normal melanogenesis? During oogenesis in both WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e, \u003cem\u003etfeb\u003c/em\u003e and \u003cem\u003etfec\u003c/em\u003e mRNA levels were low, whereas \u003cem\u003etfe3\u003c/em\u003e mRNA levels were relatively high and showed a similar trend to \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003etyrp1\u003c/em\u003e, and \u003cem\u003edct\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.N-O and Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.A-C). There were no significant differences in \u003cem\u003etfe3\u003c/em\u003e mRNA expression between WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.N). Additionally, the amino acid sequences of TFE3 and MITF are largely identical (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.D). We hypothesize that during oocyte development in \u003cem\u003eXenopus tropicalis\u003c/em\u003e, melanin deposition in the animal pole of oocytes may be regulated by one or more members of the MiT subfamily (MITF, TFEB, TFE3, and TFEC). These factors might control the expression of genes such as \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003edct\u003c/em\u003e, and \u003cem\u003etyrp1\u003c/em\u003e as needed to facilitate melanogenesis (UM2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.P). Alternatively, the MiT subfamily may regulate the transcription of these genes into mRNA, which is then stored as maternal RNA in oocytes and degraded or translated as necessary to complete melanogenesis (UM1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.P).\u003c/p\u003e \u003cp\u003eFurther examination of the mRNA expression of melanogenesis-related genes (\u003cem\u003eoca2\u003c/em\u003e, \u003cem\u003epmel\u003c/em\u003e, \u003cem\u003eslc24a5\u003c/em\u003e, \u003cem\u003eslc45a2\u003c/em\u003e, \u003cem\u003eatp7a\u003c/em\u003e, and \u003cem\u003egpnmb\u003c/em\u003e) revealed patterns similar to those of \u003cem\u003etfe3\u003c/em\u003e and \u003cem\u003etyr\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A). Specifically, during stages 1 to 3 of oocyte development, these genes maintained relatively high expression levels, which significantly decreased during stages 4 to 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A). In contrast, the mRNA expression of genes involved in the synthesis of pterinosomes in xanthophores and guanine platelet crystals in iridophores did not exhibit the same pattern as \u003cem\u003etfe3\u003c/em\u003e and \u003cem\u003etyr\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.B-C). This suggests that the synthesis of pigments related to pterinosomes and guanine platelet crystals in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes is likely not regulated by Tfe3. The expression trend of melanogenesis-related genes in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes closely mirrored that of \u003cem\u003etfe3\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A), indicating a potential regulatory role of Tfe3 in melanogenesis-related gene expression. However, further experimental validation is required. Overall, melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes appears to depend on Tyr rather than Mitf, challenging the previous understanding of Mitf as the master regulator of melanocyte development and melanogenesis and suggesting a different role for \u003cem\u003emitf\u003c/em\u003e in oocyte development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOther significant molecular features during oocyte development in\u003c/b\u003e \u003cb\u003eXenopus tropicalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTranscriptome data from WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes were further analyzed to delineate key molecular features during oocyte development. Genes were selected based on a screening criterion of P-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and FPKM\u0026thinsp;\u0026ge;\u0026thinsp;1, with log2FC ranked in descending order, revealing that the top genes predominantly related to mitochondria (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The expression patterns of these mitochondrial-related genes' mRNAs were consistent between WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes, showing no significant differences between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.A-B and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Specifically, expression levels were low during stages 1 and 2, increased significantly by stage 3, peaked at stage 4, and although slightly reduced by stage 6, remained relatively high.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the development of \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes, the expression levels of four mitochondrial-related genes, \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, and \u003cem\u003end3\u003c/em\u003e, are notably high (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.A-B and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Specifically, \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, and \u003cem\u003ecox3\u003c/em\u003e encode subunits 1, 2, and 3 of cytochrome c oxidase (complex IV), respectively (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Subunits 1 and 2 form the catalytic core of complex IV, with subunit 2 transferring electrons from cytochrome c to the active center of subunit 1 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Subunit 3, though not part of the catalytic core, stabilizes the structure and function of complex IV (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Cytochrome c oxidase, located on the inner mitochondrial membrane, is the terminal enzyme of the mitochondrial electron transport chain. Its primary function is to catalyze the transfer of electrons from cytochrome c to oxygen, producing water and simultaneously pumping protons from the mitochondrial matrix into the intermembrane space (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). This creates a transmembrane electrochemical gradient (proton gradient) that drives ATP synthesis. The \u003cem\u003end3\u003c/em\u003e gene encodes subunit 3 of NADH dehydrogenase (complex I), the first complex in the mitochondrial electron transport chain (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Complex I transfers hydrogen atoms and electrons from NADH to coenzyme Q on the inner mitochondrial membrane (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). This process also involves the pumping of protons from the mitochondrial matrix into the intermembrane space, contributing to the proton gradient for ATP synthesis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Therefore, during development to stage 3, \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes undergo extensive mitochondrial biogenesis and ATP production, indicating a high demand for energy as they progress from stage 3 to mature oocytes. Among the key genes regulating mitochondrial biogenesis and functional homeostasis, \u003cem\u003emfn1\u003c/em\u003e, \u003cem\u003emfn2\u003c/em\u003e, and \u003cem\u003etfam\u003c/em\u003e mRNA levels remain relatively high throughout all stages of \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocyte development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.C-D and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These findings indicate that extensive mitochondrial biogenesis is crucial for oocyte development in \u003cem\u003eXenopus tropicalis\u003c/em\u003e. Additionally, the data suggest that the formation of the mitochondrial cloud in these oocytes is tightly regulated by a genetic network(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Further elucidation of the mechanisms and roles of mitochondrial cloud formation during oocyte development is therefore of significant importance(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe formation of the mitochondrial cloud is a significant feature during oocyte development, alongside the notable formation of the zona pellucida(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The zona pellucida is a transparent, tough, and light-permeable membrane surrounding the oocyte, primarily composed of several glycoproteins forming a clear extracellular matrix(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). This structure encases the oocyte, playing crucial roles in protection, fertilization regulation, and embryo development. The main components of the zona pellucida are glycoproteins, including ZP1, ZP2, ZP3, and ZP4(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). We examined the expression of homologous genes \u003cem\u003ezp2\u003c/em\u003e, \u003cem\u003ezp3.2\u003c/em\u003e, \u003cem\u003ezp4\u003c/em\u003e, and \u003cem\u003ezp4.2\u003c/em\u003e during \u003cem\u003eXenopus tropicalis\u003c/em\u003e oogenesis. The results showed that although the mRNA levels of \u003cem\u003ezp2\u003c/em\u003e, \u003cem\u003ezp3.2\u003c/em\u003e, and \u003cem\u003ezp4.2\u003c/em\u003e slightly decreased as oocyte development progressed, they remained relatively low without significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.E-J and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In contrast, the expression of \u003cem\u003ezp4\u003c/em\u003e mRNA remained high throughout oogenesis but significantly decreased, particularly from stage 4 to stage 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.E-J). Typically, the mRNAs of \u003cem\u003ezp2\u003c/em\u003e, \u003cem\u003ezp3.2\u003c/em\u003e, \u003cem\u003ezp4\u003c/em\u003e, and \u003cem\u003ezp4.2\u003c/em\u003e are transcribed in follicular cells, translated into corresponding proteins, and subsequently secreted around the oocyte to contribute to the formation of the zona pellucida(\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, our transcriptome sequencing detected a substantial presence of \u003cem\u003ezp2\u003c/em\u003e, \u003cem\u003ezp3.2\u003c/em\u003e, \u003cem\u003ezp4\u003c/em\u003e, and \u003cem\u003ezp4.2\u003c/em\u003e transcripts within the oocyte itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.E-J), indicating that the oocyte might also express these mRNAs during oogenesis. This finding warrants further experimental validation. Particularly, the changes in \u003cem\u003ezp4\u003c/em\u003e mRNA expression and its relationship with the acrosome reaction merit further investigation(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe examined the expression of genes associated with acentriolar spindle assembly during meiosis. The mRNA levels of \u003cem\u003epcnt\u003c/em\u003e and \u003cem\u003etacc3\u003c/em\u003e increased progressively during oogenesis, with \u003cem\u003etacc3\u003c/em\u003e mRNA showing relatively high levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.K-L and Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Pericentrin (PCNT) plays a crucial role in oocyte meiosis, being involved in the localization of acentriolar microtubule-organizing centers (aMTOCs), spindle assembly, and chromosome segregation (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). TACC3 is essential for the localization and function of aMTOCs during meiosis. It promotes microtubule nucleation and stability by interacting with microtubule-associated proteins such as chTOG (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In \u003cem\u003eXenopus tropicalis\u003c/em\u003e, the increasing expression levels of \u003cem\u003epcnt\u003c/em\u003e and \u003cem\u003etacc3\u003c/em\u003e mRNA during oogenesis suggest that these genes might play a regulatory role in meiosis.\u003c/p\u003e \u003cp\u003eAnalysis of transcriptome data from WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes reveals that the molecular characteristics associated with mitochondrial cloud formation are the most prominent during oocyte development. This finding suggests that the mitochondrial cloud is crucial for oocyte maturation. Similar subcellular structures have been observed in the oocytes of various vertebrates, including humans, mice, and zebrafish, indicating evolutionary conservation(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Thus, the \u003cem\u003eXenopus tropicalis\u003c/em\u003e mitochondrial cloud serves as an excellent model for studying its role and mechanisms in oocyte development. Furthermore, the expression patterns of zona pellucida proteins (\u003cem\u003ezp2\u003c/em\u003e, \u003cem\u003ezp3.2\u003c/em\u003e, \u003cem\u003ezp4\u003c/em\u003e, and \u003cem\u003ezp4.2\u003c/em\u003e) and meiosis-related genes (\u003cem\u003epcnt\u003c/em\u003e and \u003cem\u003etacc3\u003c/em\u003e) underscore the need to investigate their roles in oocyte development. Therefore, using \u003cem\u003eXenopus tropicalis\u003c/em\u003e as a model organism to study oogenesis is crucial for understanding reproductive biology.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eThe deposition of animal pole pigment in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes is crucial for their development, influencing oocyte polarity, growth, and subsequent embryonic development. In this study, we disrupted the bHLH-LZ domain of Mitf in \u003cem\u003eXenopus tropicalis\u003c/em\u003e, resulting in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e frogs that lack melanocytes and show significantly reduced expression of key melanogenesis genes, indicating a loss of Mitf activity. However, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e oocytes still regulate \u003cem\u003etyr\u003c/em\u003e mRNA expression to complete melanogenesis, leading to melanin deposition at the animal pole. In contrast, \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e oocytes show no melanin deposition at the animal pole. Thus, melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes depends on Tyr rather than Mitf, potentially due to other MiT subfamily factors regulating the expression of \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003edct\u003c/em\u003e, and \u003cem\u003etyrp1\u003c/em\u003e during oogenesis. We also found that mitochondrial cloud formation represents the most significant molecular change during oocyte development, alongside the expression patterns of zona pellucida proteins and meiosis-related genes such as \u003cem\u003epcnt\u003c/em\u003e and \u003cem\u003etacc3\u003c/em\u003e. These findings suggest that further elucidation of the Tyr-dependent, Mitf-independent mechanisms of animal pole pigment deposition will enhance our understanding of melanogenesis and oocyte development. Additionally, \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes will serve as an effective model for studying the mitochondrial cloud, zona pellucida, meiosis, oogenesis, and reproductive medicine.\u003c/p\u003e \u003cp\u003eThe MiT family, a group of bHLH-Zip transcription factors, primarily includes MITF, TFEB, TFEC, and TFE3(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). These transcription factors typically bind to E-box sequences (CANNTG) via their bHLH-Zip domain to regulate target gene transcription. They can form homodimers (e.g., MITF-MITF) or heterodimers (e.g., MITF-TFEB), enhancing their DNA-binding ability and specificity(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Consequently, they play crucial roles in gene expression, cell differentiation, metabolism, and autophagy. Tfec plays a direct role in regulating zebrafish iridophore development starting from the multipotent pigment cell progenitor stage(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). It is also essential for iridophore development in a lizard model(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Therefore, it is hypothesized that Tfec regulates melanocyte development by influencing the differentiation of multipotent pigment cell progenitors, thereby affecting melanogenesis.\u003c/p\u003e \u003cp\u003eAlthough TFEB shares conserved domains with MITF, there are few reports of TFEB directly regulating melanogenesis(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). However, TFEB may influence melanogenesis through autophagy and lysosomal biogenesis(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Research indicates that MITF induction of UVRAG is crucial for UV-induced tanning, while inactivation of TFEB or TFE3 has minimal effect on UVRAG expression following α-MSH stimulation(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). This implies that TFEB and TFE3 likely do not regulate melanogenesis via the α-MSH pathway, but confirms UVRAG's role in melanogenesis regulation. UVRAG, a multifunctional protein, participates in autophagy, endocytosis, and DNA damage repair(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). In melanogenesis, UVRAG maintains the localization and stability of BLOC-1 to facilitate melanogenic cargo sorting and delivery(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). TFEB, a pivotal transcription factor in autophagy and lysosomal biogenesis, activates autophagy-related genes by binding to E-box sequences, crucial for autophagosome formation, elongation, and maturation(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). UVRAG interacts with the class C Vps complex, a key component of the endosomal fusion machinery, forming the UVRAG-class-C-Vps complex. This interaction stimulates RAB7 GTPase activity and promotes the fusion of autophagosomes with late endosomes/lysosomes, enhancing the delivery and degradation of autophagic cargo and accelerating endosome\u0026ndash;endosome fusion, leading to rapid degradation of endocytic cargo(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Therefore, TFEB may impact melanogenesis by modulating the interaction between UVRAG and BLOC-1, which is influenced by the stability of the UVRAG-class-C-Vps complex regulated by autophagosomes(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough TFE3 can\u0026rsquo;t regulate melanogenesis via the α-MSH signaling pathway(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), Tfe3a can restore melanophore development at a very low frequency in zebrafish(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Additionally, TFE3 interacts with p300, further participating in melanogenesis-related signaling pathways(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Studies have shown that Tfe3 can activate the expression of Tyr and Tyrp1(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Therefore, overall, although Tfeb, Tfec, and Tfe3 may be involved in the regulation of oocyte melanogenesis, considering that Tfe3 has a higher expression level in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes compared to the very low levels of Tfeb and Tfec, and considering the evidence that Tfe3 regulates Tyr and Tyrp1 expression, we hypothesize that Tfe3, rather than Mitf, regulates melanogenesis-related gene expression during oogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e. This regulation results in melanin deposition at the animal pole of the oocyte. Further experimental data elucidating the molecular mechanisms of melanin deposition in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes will enhance research in oogenesis and reproductive medicine.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cp\u003e \u003cb\u003eXenopus tropicalis\u003c/b\u003e \u003cb\u003emaintenance and husbandry\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAdult \u003cem\u003eXenopus tropicalis\u003c/em\u003e frogs were obtained from Nasco (Fort Atkinson, WI, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.enasco.com\u003c/span\u003e\u003cspan address=\"http://www.enasco.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The maintenance and husbandry of the frogs, as well as the procurement of their oocytes, adhered to established methods(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). All experiments involving \u003cem\u003eXenopus tropicalis\u003c/em\u003e were approved by the Institutional Animal Care and Use Committee of the Southern University of Science and Technology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe procurement of\u003c/b\u003e \u003cb\u003eXenopus tropicalis\u003c/b\u003e \u003cb\u003eoocytes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe establishment of \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e lines has been reported in our previous studies(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The method for collecting oocytes from WT, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e was slightly modified from the protocol described in the literature(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). First, healthy adult WT, \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e were selected. To reduce stress, the frogs were anesthetized with 0.2% MS-222 before the procedure. The abdomen was disinfected with 70% ethanol, and a small incision (approximately 1\u0026ndash;2 cm) was made in the center of the abdomen, taking care not to cut too deeply to avoid damaging internal organs. The ovaries were carefully removed through the incision, and a portion of the ovarian tissue was gently excised using scissors or forceps. The excised ovarian tissue was placed in a dish containing cold 1\u0026times; MBS solution to prevent damage to the oocytes. The ovarian tissue was then repeatedly washed in 1\u0026times; MBS solution to remove blood and debris. After washing, the ovarian tissue was transferred to 1\u0026times; MBS solution containing 1.5 mg/mL collagenase and incubated at 25\u0026deg;C for 1\u0026ndash;5 hours with gentle agitation to facilitate the release of oocytes from the ovarian tissue. The process was monitored under a stereomicroscope to ensure that the follicle cells were completely separated from the oocytes. Following incubation, the oocytes were washed multiple times with fresh 1\u0026times; MBS solution to remove residual collagenase and debris. The oocytes were then observed under a stereomicroscope, and oocytes at various developmental stages were selected.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq library preparation and RNA-seq analysis\u003c/h2\u003e \u003cp\u003eOocytes were collected from WT and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e adult frogs, with each group comprising two replicates. Oocytes were disrupted using a disposable syringe until completely lysed, and total RNA was extracted following the manufacturer\u0026rsquo;s instructions for the TransZol Up lysis reagent (ET111-01, TransGen Biotech). The extracted RNA samples were submitted to Novogene for RNA-seq library preparation and sequencing. RNA-seq analysis was conducted by Wuhan Frasergen Information Co., Ltd. The previously published protocol guided the RNA-seq data analysis(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Briefly, SOAPnuke software (v2.1.0) was used to filter raw reads to obtain clean reads. Clean reads were aligned to the \u003cem\u003eXenopus tropicalis\u003c/em\u003e reference genome UCB_Xtro_10.0 using HISAT2. Bowtie2 software then mapped the quality-controlled sequences to the reference transcriptome. RSEM analyzed the Bowtie2 alignment results to determine the number of reads mapped to each transcript and calculate FPKM (Fragments Per Kilobase per Million bases) values. Differential expression analysis was performed using DESeq2. RNA-seq data has been publicly deposited to Genome Sequence Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/gsa/\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/gsa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the accession number CRA017994 (Reviewers can obtain the RNA-seq data through this link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/gsa/s/1WKfk9MH\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/gsa/s/1WKfk9MH\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-Eosin staining, transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eThe samples underwent histological analysis, starting with fixation in FAS eyeball fixative (Servicebio, Wuhan, China) at room temperature for 24 hours. The fixed samples were then dehydrated using graded ethanol (75%, 85%, 95%, and 100%), followed by replacement with xylene and embedding in paraffin wax. Sections with a thickness of 6 \u0026micro;m were prepared from the embedded tissues and subsequently dewaxed in xylene. Rehydration was carried out using graded ethanol concentrations (100%, 95%, and 70%) before staining with the hematoxylin-eosin staining kit (Baso, Zhuhai, China) and immunofluorescent staining. Hematoxylin-eosin-stained results were captured using an Olympus BX53 upright microscope (Olympus, Japan).\u003c/p\u003e \u003cp\u003eOur previously published protocol guided the TEM sample preparation(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, \u003cem\u003eXenopus tropicalis\u003c/em\u003e tissue samples, sized 1 mm \u0026times; 1 mm, were fixed overnight at 4\u0026deg;C in 2% glutaraldehyde. Oocytes were also fixed overnight at 4\u0026deg;C in 2% glutaraldehyde. Following fixation, the samples underwent four 10-minute washes with 10 mM PBS solution. Subsequently, the samples were fixed with 1% osmium tetroxide at room temperature for 3 hours. The samples were then washed twice with 10 mM PBS solution for 10 minutes each, followed by two washes with ddH\u003csub\u003e2\u003c/sub\u003eO for 10 minutes each. After rinsing with ddH\u003csub\u003e2\u003c/sub\u003eO, the samples were treated with a 2% uranyl acetate solution at room temperature for 2 hours or overnight at 4\u0026deg;C. Following this, the samples were washed four or more times with ddH\u003csub\u003e2\u003c/sub\u003eO for 10 minutes each until the rinsed water was clear. Post-rinsing, the samples were dehydrated using a graded acetone series: 30% acetone once, 50% acetone once, 75% acetone once, and 100% acetone twice, each for 10 minutes. Simultaneously with the gradient dehydration, epoxy resin (9.8 g), DDSA (5.6 g), NMH (4.6 g), and DMP-30 (0.28 mL) were thoroughly mixed at room temperature using a rotary mixer for at least 4 hours to prepare a 100% resin mixture. Then, at room temperature, the samples were treated with 25% resin-acetone once, 50% resin-acetone once, 75% resin-acetone once, and 100% resin once, each for 1.5-2 hours. Following these steps, new 100% resin was added, and the samples were left overnight at 4\u0026deg;C. Unused 100% resin was sealed with parafilm and stored at 4\u0026deg;C for later use. The next day, the samples were embedded in new 100% resin. Subsequently, the embedded samples were baked in a 60\u0026deg;C oven for 2 days to complete the preparation of TEM samples for ultrathin sectioning. Finally, the prepared TEM samples were sent to Wuhan Servicebio Technology Co., Ltd. for ultrathin sectioning, TEM observation, and photography.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study data are included in the article and/or supplemental information. No other scripts and software were used other than those mentioned in the Methods section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Nos.\u003c/p\u003e\n\u003cp\u003e32300659, 92169119, 82070420), Shenzhen Science and Technology Innovation Commission Project (No. JCYJ20230807143302004), Shanghai Science and Technology Innovation Action Plan (No. 22N31900800), Shenzhen Clinical Research Center for Emerging Infectious Diseases (No. LCYSSQ20220823091203007), Shenzhen High-level Hospital Construction Fund (No. XKJS-CRGRK-008), and National Key Research and Development Program of China (Nos. 2022YFC2304401，2022YFC2304402).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.R. and H.L. and Y.C. and W.L. conceived the project. W.L. and J.H. and J.S. performed the experiments and analyzed the data together with S.Y. and H.L. and J.D. and X.F.. S.H. and W.C. contributed TEM data collation and analysis. R.R. and H.Y. wrote the manuscript with input from all the authors. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the other members of our team for their help to the project.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eF. S\u0026aacute;nchez, J. Smitz, Molecular control of oogenesis. \u003cem\u003eBiochimica et Biophysica Acta (BBA)-Molecular Basis of Disease\u003c/em\u003e \u003cstrong\u003e1822\u003c/strong\u003e, 1896-1912 (2012).\u003c/li\u003e\n\u003cli\u003eM. J. 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Shi\u003cem\u003e et al.\u003c/em\u003e, Activation of P53 pathway contributes to Xenopus hybrid inviability. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e120\u003c/strong\u003e, e2303698120 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oogenesis, melanogenesis, Xenopus tropicalis, mitochondrial cloud ","lastPublishedDoi":"10.21203/rs.3.rs-4807093/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4807093/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMelanin pigmentation in oocytes is a critical feature for both the aesthetic 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. Here, we compare the oocytes of wild-type, \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eXenopus tropicalis\u003c/em\u003e and found that \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e oocytes exhibit normal melanin deposition at the animal pole, whereas \u003cem\u003etyr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e oocytes show no melanin deposition at this site. Transmission electron microscopy confirmed that melanogenesis in \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e oocytes proceeds normally, similar to wild-type oocytes. Transcriptomic analysis revealed that \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e oocytes regulate the expression of melanogenesis-related genes to complete melanogenesis. Additionally, in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes, the expression of the MiT subfamily factor \u003cem\u003etfe3\u003c/em\u003e is relatively high, while \u003cem\u003etfeb\u003c/em\u003e, \u003cem\u003emitf\u003c/em\u003e, and \u003cem\u003etfec\u003c/em\u003e levels are extremely low. The expression pattern of \u003cem\u003etfe3\u003c/em\u003e is similar to that of \u003cem\u003etyr\u003c/em\u003e and other melanogenesis-related genes. Thus, melanogenesis in \u003cem\u003eXenopus tropicalis\u003c/em\u003e oocytes is dependent on Tyr rather than Mitf, possibly due to the regulation of \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003edct\u003c/em\u003e, and \u003cem\u003etyrp1\u003c/em\u003e by other MiT subfamily factors such as \u003cem\u003etfe3\u003c/em\u003e. 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. Overall, these findings suggest that further elucidation of Tyr-dependent, Mitf-independent mechanisms of melanin deposition at the animal pole will enhance our understanding of melanogenesis and Oogenesis.\u003c/p\u003e","manuscriptTitle":"Dispensable role of mitf in melanogenesis of Xenopus tropicalis oocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 17:09:57","doi":"10.21203/rs.3.rs-4807093/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-23T16:03:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-23T15:54:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-14T02:32:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-13T08:14:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336003924098344149615271189649483820534","date":"2024-09-07T15:26:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229298053771266893515955081025690259960","date":"2024-09-06T14:39:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306599962685183233811385483646810927372","date":"2024-09-06T01:20:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-01T09:50:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T07:54:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-29T08:49:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2024-07-26T09:39:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ef84f22-8457-4d76-bbb5-372d2d430594","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-18T01:08:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 17:09:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4807093","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4807093","identity":"rs-4807093","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}