Manganese exposure induces cellular aggregates and the accumulation of β-catenin in skin of zebrafish embryos

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Here we studied the effects of Mn in zebrafish, at the cellular level, due to the transparent nature of zebrafish larvae that enables a powerful analysis under the light microscope. The collection of our results shows that environmental concentrations of 0.5 mg/L affect swimbladder inflation; at concentration of 50 mg/L and 100 mg/L Mn (i) induces alterations in viability, swim bladder, heart, and size of zebrafish larvae, (ii) induces an increase in melanocyte area and the formation of cellular aggregates in the skin, and (iii) induces an accumulation of β-catenin in mesenchymal cells in the caudal fin of zebrafish larvae. Our data suggest that increased levels of Mn induce cell aggregate formation in the skin and the presence of more melanocytes in the zebrafish caudal fin. Interestingly, the adhesion protein β-catenin was activated in mesenchymal cells near the cell aggregates. These results open important new questions on the role of Mn toxicity on cellular organization and β-catenin responses in fishes. Mn manganese zebrafish fin epithelial cells skin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Manganese (Mn) is a trace metal and a cofactor to enzymes with essential biological functions in nearly all human tissues, including metabolism, growth, replication, and differentiation (Aschner & Erikson, 2017 ; Erikson & Aschner, 2019 ). However, exposure to high levels of Mn can lead to severe pathologies, where neurological disorders are the most evident (Altenhofen et al., 2017 ; Huang et al., 2011 ; O’Neal et al., 2014 ; Yoon et al., 2011 ). Importantly, there is still a void in knowledge regarding the effects of Mn in tissues other than the nervous system, particularly using analysis at the molecular and cellular levels. One of the most promising vertebrate animal models for studies of human-related diseases is the zebrafish ( Danio rerio ). The many advantages of zebrafish include its well characterized genome and its excellent optical clarity, which is well suited for studies on early stages of embryonic development. Zebrafish has been widely used to investigate the effects of Mn, including a manganism model (Altenhofen et al., 2017 ; Arndt et al., 2014 ; Bakthavatsalam et al., 2014 ; Herman et al., 2021 ; Hernández et al., 2015 ; Marins et al., 2019 ; Tu et al., 2017 ; Zimmermann Prado Rodrigues et al., 2020). It has been shown that Mn content levels increase in the zebrafish after 5 days-post-fertilization (dpf), when they start to feed from external sources (Thomason et al., 2017 ). Interestingly, at this developmental stage the larvae increase sensitivity to Mn exposure (Altenhofen et al., 2017 ; Bakthavatsalam et al., 2014 ; Hernández et al., 2015 ; Tu et al., 2017 ). Nevertheless, none of these studies analyzed the cellular effects of Mn during zebrafish early developmental stages. Although zebrafish get more sensitive after 5 dpf, we have used early stages until 96hpf larvae to investigate in more detail the complexity of larvae phenotypes exposed to Mn. We found that at concentrations of 0.5 mg/L, 5 times above the acceptable for potable water in Brazil (Conama, 2005 ), detected in polluted environments (da Silva, 2015 ; Francisco et al., 2019 ; Queiroz et al., 2021 ), the zebrafish embryos presented a non-inflated swim bladder. Moreover, we describe interesting phenotypes besides the neurological effects in the literature, such as the formation of skin aggregates, cellular infiltration and increased number of melanocytes. Therefore, we aimed to analyze the cellular effects of Mn during zebrafish development using high quality light microscopy and confocal fluorescence microscopy. Mn induced an increased expression of the signaling protein β-catenin on cells beneath the skin aggregates with mesenchymal morphology. Our data suggest a disruptive effect of Mn on skin cellular organization and the regulation of Wnt/β-catenin on mesenchymal cells that were not previously described in the literature. 2. Materials And Methods 2.1 Zebrafish husbandry and embryo exposure to Mn Zebrafish ( Danio rerio ) were maintained in aquaria with recirculating water system at 28 ± 1°C on a 14:10 light/dark cycle in animal facility at the Institute of Biodiversity and Sustainability (NUPEM) of the Federal University of Rio de Janeiro, Macaé-RJ, Brazil according to (Westerfield, 2007 ). Animals were handled and experimented according to Institutional Animal Care and Use Committee protocols under the number 067/17. Embryos and larvae were collected and treated at an early stage with increasing concentrations of MnCl2 (0.5 mg/L, 5 mg/L, 50 mg/L, 100 mg/L and 500 mg/L) up to 96 hours post fertilization (hpf). 2.2 Sudan Black labeling Sudan Black labeling protocol was modified from previous studies (Le Guyader et al., 2008 ; Sheehan & Storey, 1947 ; Walters et al., 2009 ). Control and Mn-treated zebrafish larvae at 96-hpf stages were fixed in 4% paraformaldehyde overnight. Next, larvae were washed five times with PBS and incubated with 0.3% Sudan Black Solution (dissolved in ethanol, phenol and Na2HPO.12H2O) for 20 min. Then, larvae were washed five times with 70% ethanol and incubated for 15 min with 1% KOH, 1%H2O2 and EtOH 70%. Larvae were incubated for 10 min with PBS containing 0.1% Tween 20 and 50% ethanol, followed by 10 min with 30% ethanol and digital images of larvae were obtained in a light microscope. 2.3 Antibodies and probes Rabbit polyclonal antibody against beta-catenin (code # C-2206) was from Sigma-Aldrich (USA). DNA-binding probe 4,6-Diamino-2-phenylindole dihydrochloride (DAPI) and Alexa Fluor 546-goat anti-rabbit IgG antibody was from Molecular Probes (USA). 2.4 Immunofluorescence Dechorionated zebrafish larvae at 96 hpf were fixed in 4% paraformaldehyde in PBS for 1h at room temperature. Larvae were then permeabilized with 0.5% Triton-X 100 in PBS (PBS/T) three times for 30 min and incubated overnight at 4°C with primary antibodies (all diluted 1:100 in PBS/T). Then, larvae were washed for 30 min with PBS/T and incubated for 1 hour at 37º C with Alexa Fluor-conjugated secondary antibodies (all diluted 1:200 in PBS/T). Nuclei were labeled with 0.1 µg/mL of DAPI in 0.9% NaCl. Larvae were mounted on #1.5 24 x 60-mm glass coverslips (with spacers) using Prolong Gold (Molecular Probes). Experiments with zebrafish larvae were repeated four times. 2.5 Fluorescence image acquisition and processing Zebrafish larvae were examined in an Axiovert 100 microscope (Carl Zeiss, Germany) coupled to an Olympus DP71 high-resolution camera, and in a DSU Spinning Disk confocal scanner mounted on an inverted fluorescent microscope (Olympus, Japan) and TCS SPE confocal (Leica). For live imaging, live embryos were mounted in 1% methyl cellulose and bright-field microscopy was performed on a Leica stereomicroscope (Leica, Germany). Image processing (brightness, contrast adjustments and area quantification) was performed using Fiji software (Schindelin et al., 2012 ) and figure panels were mounted with Adobe Photoshop software (Adobe Systems Inc., USA). 2.6 Statistical analysis Statistical analysis was carried out using the GraphPad Prism software version 8. The results are expressed as mean ± SEM. The groups were performed with at least three independent experiments (N = 3), where ***p < 0.001, **p < 0.01 and *p < 0.05 vs. control group. Statistical analysis was performed using one-way ANOVA. 3. Results And Discussion 3.1 Analysis of mortality, hatching and alterations in swim bladder and heart after in zebrafish larvae after manganese treatment To study the effects of manganese (Mn) in zebrafish embryos, we first analyzed the mortality rate ( Supplemental Fig. 1 ) of zebrafish larvae exposed to different concentrations of Mn (0.5, 5, 50, 100 and 500 mg/L). The mortality rate for all concentrations until 96 hpf were below 10% ( Supplemental Fig. 1 ). It is comparable to what has been described in the literature, where the mortality to Mn exposure increase after 96 hpf (Altenhofen et al., 2017 ; Bakthavatsalam et al., 2014 ; Hernández et al., 2015 ; Tu et al., 2017 ). However hatching rate at 500 mg/L was significantly impaired with strong morphological retardation of the embryos at 96 hpf (Fig. 1 A and 1 I). It is possible that non-hatching embryos exposed to 500 mg/L (9 mM) would not survive after 5dpf. If we consider the LC50 of embryo-larvae exposed to MnCl2 for 48h after 3.5 dpf the LC50 was of 2.0 mM (Bakthavatsalam et al., 2014 ). In another study, the embryos exposed from 48 hpf-120 hpf had a LC50 range of 6.0-10mM (Hernández et al., 2015 ). Regarding the morphological phenotypes, apart from neurological and craniofacial morphological malformations observed in the literature (Altenhofen et al., 2017 ; Bakthavatsalam et al., 2014 ; Tu et al., 2017 ), we found alterations in swim bladder, heart and cell aggregates on the skin of zebrafish larvae. Swimbladder fails to inflate in 60% of the larvae in 0.5 mg/L and 77% in 5 mg/L (Fig. 1 ). While all embryos exposed to 50, 100 and 500 mg/L of Mn presented influence in swim bladder inflation (Fig. 1 C-I). The Mn concentration exposure started with 0.5 mg/L that is similar to what has been found in polluted environmental samples (da Silva, 2015 ; Francisco et al., 2019 ; Queiroz et al., 2021 ) and is above the 0.1 mg/L permitted in potable water in Brazil (Conama, 2005 ). The swim bladder inflates at 4.5 dpf when the larvae have 4.4mm (Lindsey et al., 2010 ; Winata et al., 2009 ). It is a gas-filled organ that is important for fish buoyancy and has similarities to tetrapod lungs (Lindsey et al., 2010 ; Winata et al., 2009 ). This data reinforces the importance of keeping the Mn levels in the water below 0.5 mg/L. Another phenotype noticed in zebrafish embryos was cardiac edema, which was detected in 30% of the larvae in 50 mg/L, 50% in 100 mg/L and 25% in 500 mg/L (Fig. 1 F- 1 I). Indeed, Mn has been shown to cause heart disturbance in many vertebrates including humans (Créton et al., 1998 ; Jiang & Zheng, 2005 ). Workers exposed to Mn present higher alterations in electrocardiogram (ECG) than control group, caused by different types of cardiac alterations (Jiang & Zheng, 2005 ). In zebrafish embryos, Mn induce calcium decrease (Hernández et al., 2015 ) and pericardial edema (Tu et al., 2017 ). Besides calcium dysregulation leads to failure of heart development in zebrafish (Créton et al., 1998 ), calcium itself has important roles along zebrafish development (Webb & Miller, 2007 ). In case calcium signaling disruption in the embryo would be proportional to Mn concentration, it could mean that as higher the concentration of Mn, more calcium imbalance would be generated. This could be one of the explanations why the embryos exposed to 500 mg/L had a lower percentage of heart edema than the ones exposed to 100 mg/L, including its strong impairment in development (Fig. 1 I). However, this should be further investigated in future work. 3.2 Manganese treatment induces an increase in melanocyte area and the formation of skin aggregates in zebrafish larvae Larvae treated with 500 mg/L Mn had severe development impairment, therefore, we carried out the detailed phenotype investigation of 50 and 100mg/L Mn treated embryos. At 96hpf, the larvae had a significant decrease in the total length after 100 mg/L of Mn (6% decrease compared to untreated larvae) ( Supplemental Fig. 2 ). Further analysis of zebrafish larvae caudal fin size under the light microscope, exhibited an extended area of melanocyte occupation after Mn exposure. Quantification of the melanocyte area showed an 50% increase after 50 mg/L Mn, whereas no change was observed after 100 mg/L Mn (Fig. 2 ). Interestingly, Mn has a high affinity for melanin tissues in vitro and in vivo (Lydén et al., 1984 ) and accumulate in specific regions of the brain, especially in pigmented neurons of substantia nigra (O’Neal & Zheng, 2015 ). The toxicity of Mn in the nervous system has been widely described, but little data is related to skin melanocytes. There is a poster abstract reporting that human melanocytes viability decreases in a dose dependent manner upon Mn exposure (Kim et al., 2005 ). It remains to be investigated the response of melanocytes to Mn, related to growth and lethality. Mn in the brain induce mitochondrial impairment that lead to a series of events ending in apoptosis induction (reviewed in Pajarillo et al., 2021 ). Would it be the same for other melanin-containing cells? We analyzed another group of cells that presented abnormal behavior that were not previously described in the literature. One of the unexpected phenotypes found was the presence of skin aggregates on the larvae. We found a 1.5 increase in skin aggregates in the larvae treated with 50 mg/L Mn (Fig. 3 ). The cellular aggregates were clearly visible under the light microscope without any kind of labeling or staining. Quantification of the size of the cellular aggregates showed that they have an average size of 1.53 mm 2 after 50 mg/L of Mn and 0.43 mm 2 in 100 mg/L Mn (Fig. 3 ). Previous reports of Mn toxicity in zebrafish larvae did not cite those skin aggregates. The tail fin is a very fragile structure. Possibly the larvae preparation used for analyses, smashed the tail fins and this phenotype was overlooked (Bakthavatsalam et al., 2014 ; Hernández et al., 2015 ; Tu et al., 2017 ). It is known that Mn deficient diets in young men lead to skin rashes (Friedman et al., 1987 ). On the other hand, patients exposed to MnCl 2 presented irritant reactions and it was cytotoxic to keratinocytes cell culture (Shallcross et al., 2014 ), suggesting an important role of Mn in skin homeostasis. Ionic imbalance in zebrafish atpase1b1a mutant, a mutation in Na,K-ATPase β1-subunit, demonstrated the formation of skin aggregates derived from the proliferation of basal keratinocytes with metastasis capacity (Hatzold et al., 2016 ). The skin aggregates after Mn exposure were morphologically very similar to atpase1b1a mutant. Mn affect the expression and/or function of other atpases as ATP13A2 and ATP2C1 and calcium balance (Gavin et al., 1990 ; Tan et al., 2011 ; Xia et al., 2017 ). The aggregates from atpase1b1a mutants produced metalloproteinase-9 (MMP) capable of degrade basement membrane and is a malignancy marker (Hatzold et al., 2016 ). Mn induces MMP9 expression in human keratinocytes (Chebassier et al., 2004 ) and rat astrocytes (Latronico et al., 2013 ). Future experiments should characterize if the skin aggregates have similar malignant characteristics as the ones formed by ionic disturbances. Another important characteristic of Mn toxicity is the induction of an inflammatory environment, inducing interleukin-6 and tumor necrosis factor α (Kobayashi et al., 2007 ; Milatovic et al., 2009 ; Shallcross et al., 2014 ). To further analyze for cell infiltrates possibly induced by inflammation, we have stained 96 hpf larvae with Sudan Black (SB) (Le Guyader et al., 2008 ; Sheehan & Storey, 1947 ). SB stain rich lipid organelles of granulocytes and has been characterized in zebrafish to stain myeloperoxidase-positive granules of neutrophils (Le Guyader et al., 2008 ). SB stained the cellular infiltrates in the caudal fin of zebrafish larvae, and the area of aggregates had SB-positive cells after Mn treatment, as compared to untreated larvae (Fig. 4 ), indicating an inflammatory response. 3.3 Manganese treatment induces an accumulation of beta-catenin in aggregates in the caudal fin of zebrafish larvae The disturbances in cell organization in the caudal fin of zebrafish larvae after Mn treatment raise the idea to analyze the distribution of proteins related to tissue structure. β-catenin is a structural and signaling protein that is essential for the generation and maintenance of epithelial tissues organization (van der Wal & van Amerongen, 2020 ). β-catenin localization in the nucleus indicates activation of the canonical Wnt/β-catenin pathway (van der Wal & van Amerongen, 2020 ). Immunofluorescence labeling of β-catenin and the nuclear dye DAPI showed β-catenin in 96 hpf larvae control were more diffuse along the skin cells (Fig. 5 A-C). However, in Mn exposed embryos, β-catenin was concentrated in cells beneath the caudal fin aggregates; and not in the cell aggregates (Fig. 5 D-I). In higher magnification, it is possible to see that β-catenin-positive cells have an elongated morphology, resembling mesenchymal migrating cells below the skin (Fig. 5 D-F). However, the staining is not all over the dermal-mesenchymal cells that migrate into the caudal fins (Feitosa et al., 2012 ). It is possible that they could be myeloid migrating cells. Further investigation on the nature of these positive-β-catenin cells should be analyzed. However it is exciting to think that there is a cross-talk between NFκB and Wnt/β-catenin in inflammatory events, acting positively or negatively (Ma & Hottiger, 2016 ). Recent literature demonstrates that ficetin, a flavonoid, activated β-catenin in zebrafish larvae and induced melanogenesis (Molagoda et al., 2020 ) and in macrophages the β-catenin activation can inhibit the NFκB signaling, acting as an anti-inflammatory (Molagoda et al., 2021 ). This leads to future questions to be answered on the role of Mn toxicity in β-catenin activation in mesenchymal cells with the potential to act as an inflammatory mediator in skin damage. 4. Conclusions Here, we studied the effects of manganese (Mn) during zebrafish development and found that concentration of 50 and 100 mg/L of Mn (i) induces alterations in swim bladder inflation, heart, and size of zebrafish larvae, (ii) induces an increase in melanocyte area and the formation of cellular aggregates in the caudal fin, and (iii) induces an accumulation of β-catenin in mesenchymal cells in the caudal fin of zebrafish larvae. These results open important new questions on the Mn role in skin cell organization, melanocyte behavior and the multifunctional protein β-catenin in the cellular responses in zebrafish. Declarations Data Availability Statement The datasets generated for this study can be found within the manuscript figures. Ethics Statement The use of zebrafish embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro and received the approval number 067/17. Author Contributions- Consent to Participate NMF conceived the work. GG and JLO contributed to the acquisition of data. CM, GG, JLO, MC and NMF contributed to the analysis and interpretation of data. CM and NF wrote the first draft of the manuscript. All authors consent to participate, contributed to manuscript revision, read, and approved the submitted version. Consent to Publish All listed authors consent and agree with the publication of the data present within this manuscript. The authors declare that they have no conflict of interest. Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, funding number 302115/2017-0 for CM, 301443/2018-1 for MLC, 431352/2018-6 for NMF) and Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, funding number E-26/202.920/2019 for CM and E26/210.220/2018 for MLC). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Altenhofen S, Wiprich MT, Nery LR, Leite CE, Vianna MR, M. R., Bonan CD (2017) Manganese(II) chloride alters behavioral and neurochemical parameters in larvae and adult zebrafish. 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A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th Edition. University of Oregon Press, Eugene (Book) Winata CL, Korzh S, Kondrychyn I, Zheng W, Korzh V, Gong Z (2009) Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers. Dev Biol 331(2). https://doi.org/10.1016/j.ydbio.2009.04.035 Xia Z, Wei J, Li Y, Wang J, Li W, Wang K, Hong X, Zhao L, Chen C, Min J, Wang F (2017) Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis. PLoS Genet 13(7). https://doi.org/10.1371/journal.pgen.1006892 Yoon H, Kim D-S, Lee G-H, Kim K-W, Kim H-R, Chae H-J (2011) Apoptosis Induced by Manganese on Neuronal SK-N-MC Cell Line: Endoplasmic Reticulum (ER) Stress and Mitochondria Dysfunction. Environmental Health and Toxicology , 26 . https://doi.org/10.5620/eht.2011.26.e2011017 Zimmermann P, Rodrigues G, Staudt LBM, Moreira MG, dos Santos TG, de Souza MS, Lúcio CJ, Panizzon J, Kayser JM, Simões LAR, Ziulkoski AL, Bonan CD, de Oliveira DL, Gehlen G (2020) Histopathological, genotoxic, and behavioral damages induced by manganese (II) in adult zebrafish. Chemosphere , 244 . https://doi.org/10.1016/j.chemosphere.2019.125550 Supplementary Files SupplementalFigures.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2022 Reviewers invited by journal 18 May, 2022 Editor invited by journal 09 May, 2022 Editor assigned by journal 06 May, 2022 First submitted to journal 18 Apr, 2022 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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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1570773","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":107061889,"identity":"eabbef7d-5a79-46c4-a15a-078a7e443e14","order_by":0,"name":"Geyse Gomes","email":"","orcid":"","institution":"Federal University of Rio de Janeiro: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Geyse","middleName":"","lastName":"Gomes","suffix":""},{"id":107061890,"identity":"2e96d932-f932-47f2-aa15-97a8799ae08d","order_by":1,"name":"José Leonardo Oliveira","email":"","orcid":"","institution":"Federal University of Rio de Janeiro: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"Leonardo","lastName":"Oliveira","suffix":""},{"id":107061891,"identity":"09048e96-c52d-4139-bb32-46396c6e25f3","order_by":2,"name":"Manoel Luis Costa","email":"","orcid":"","institution":"Federal University of Rio de Janeiro: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manoel","middleName":"Luis","lastName":"Costa","suffix":""},{"id":107061892,"identity":"32dfe857-de6e-4c5f-a12b-3b762e120c4c","order_by":3,"name":"Claudia Mermelstein","email":"","orcid":"","institution":"Federal University of Rio de Janeiro: Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Mermelstein","suffix":""},{"id":107061893,"identity":"9bae8041-bbf9-4c14-8123-c6514cba520a","order_by":4,"name":"Natália Martins Feitosa","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9425-8018","institution":"UFRJ: Universidade Federal do Rio de Janeiro","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Natália","middleName":"Martins","lastName":"Feitosa","suffix":""}],"badges":[],"createdAt":"2022-04-19 02:36:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1570773/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1570773/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22068923,"identity":"8564cb0d-4b5e-42e0-94a5-450002c8995b","added_by":"auto","created_at":"2022-05-31 14:07:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn treatment affects hatching and induces heart, swim bladder and skin alterations.\u003c/strong\u003e Zebrafish eggs were treated with Mn at 0.5, 5, 50, 100 and 500 mg/L and analyzed under a light microscope. Only 500 mg/L of Mn showed a significant inhibition of hatching (A and I). The zebrafish larvae also showed alteration in the swim bladder after treatment with all Mn concentrations tested (B, D-G), whereas cardiac edema and skin aggregates were found only after treatment with Mn levels higher than 50 mg/L (E-H). The results are expressed as mean ± SEM. The groups were performed with n = 5, ****p \u0026lt; 0.0001, ***p \u0026lt; 0.001 and **p and *p \u0026lt; 0.05 where vs. control group. ns = non-significant. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test. Scale bar in C, H and I = 500 μm. Arrowhead = cardiac edema; arrow = swim bladder; * = blood.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/a2276efa61d17a03cd1119cd.jpg"},{"id":22067768,"identity":"f92d4b8d-6ba5-457d-9d21-317b716069c3","added_by":"auto","created_at":"2022-05-31 13:57:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn treatment increases the area of melanocytes at the zebrafish fin. \u003c/strong\u003eZebrafish larvae were treated with Mn at 50 or 100 mg/L (A-I) and analyzed under a light microscope. Mn treatment with 50 mg/L induces an increase in the area of melanocytes (J), but not with 100 mg/L (J). Scale bar in I = 100 μm. The results are expressed as mean ± SEM. The groups were performed with n = 5, where **p \u0026lt; 0.05 vs. control group. ns = non-significant. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/0169645b4d2c0ea08ef69418.jpeg"},{"id":22068339,"identity":"c6423b94-eb7b-4c1a-a626-ee342e527967","added_by":"auto","created_at":"2022-05-31 14:02:52","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn treatment induces the formation of cellular aggregates at the zebrafish fin.\u003c/strong\u003e Zebrafish larvae were treated with Mn at 50 or 100 mg/L (A-F) and analyzed under a light microscope. Quantification of the area of cellular aggregates found at zebrafish fin shows an increase after Mn treatment (D). Scale bars in E =\u0026nbsp;100 μm and F =\u0026nbsp;50 μm. The results are expressed as mean ± SEM. The groups were performed with n = 5, where **** p \u0026lt; 0.0001 and *p \u0026lt; 0.05 vs. control group. Statistical analysis was performed using one-way ANOVA followed by Dunn’s post-test.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/d2b19fc1446c7500df3c1723.jpeg"},{"id":22068338,"identity":"6b8ba14a-fbd0-46e7-b5a4-fef682d65b8c","added_by":"auto","created_at":"2022-05-31 14:02:52","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn treatment increases the number of Sudan Black-positive cells at the zebrafish fin.\u003c/strong\u003e Zebrafish larvae were treated with Mn at 50 or 100 mg/L, stained with Sudan Black and analyzed under a light microscope (A-C). Scale bar in C = 500 μm. The groups were performed with n = 5.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/e65e57c095ba82a97d86fcb8.jpeg"},{"id":22067771,"identity":"a0d2efaf-5ba1-45fd-987a-2247fba69eda","added_by":"auto","created_at":"2022-05-31 13:57:52","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":438459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMn treatment disorganizes the epithelium and induces the expression of beta-catenin.\u003c/strong\u003e Confocal analysis of zebrafish larvae at 96 hours\u003cstrong\u003e \u003c/strong\u003epost-fertilization stained with beta-catenin (red) and DAPI (blue). In the control embryo, beta-catenin is concentrated at typical intercellular adherens junctions of epithelial cells (A-C). In the 50 mg/L Mn-treated embryo (D-F), beta-catenin-positive intercellular junctions appear disorganized and into the nuclei of some mesenchymal cells. In embryos treated with 100 mg/L Mn (G-I), beta-catenin is concentrated in some areas of the epithelial tissue. Scale bar in I = 50 μm. The groups were performed with n = 5.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/03c975ece99657727cf6b79b.jpeg"},{"id":22068925,"identity":"f188673a-887a-4321-b05b-2ddfcff91215","added_by":"auto","created_at":"2022-05-31 14:07:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1052386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/1d42f887-7124-42f7-9c66-158c6a9edde9.pdf"},{"id":22067772,"identity":"49979f02-8d7a-4c17-89d1-6db53cd94c73","added_by":"auto","created_at":"2022-05-31 13:57:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":350875,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-1570773/v1/6dfcf1876587902a63f3b0c7.docx"}],"financialInterests":"","formattedTitle":"Manganese exposure induces cellular aggregates and the accumulation of β-catenin in skin of zebrafish embryos","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eManganese (Mn) is a trace metal and a cofactor to enzymes with essential biological functions in nearly all human tissues, including metabolism, growth, replication, and differentiation (Aschner \u0026amp; Erikson, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Erikson \u0026amp; Aschner, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, exposure to high levels of Mn can lead to severe pathologies, where neurological disorders are the most evident (Altenhofen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; O\u0026rsquo;Neal et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yoon et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Importantly, there is still a void in knowledge regarding the effects of Mn in tissues other than the nervous system, particularly using analysis at the molecular and cellular levels. One of the most promising vertebrate animal models for studies of human-related diseases is the zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). The many advantages of zebrafish include its well characterized genome and its excellent optical clarity, which is well suited for studies on early stages of embryonic development. Zebrafish has been widely used to investigate the effects of Mn, including a manganism model (Altenhofen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Arndt et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bakthavatsalam et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Herman et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Marins et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zimmermann Prado Rodrigues et al., 2020). It has been shown that Mn content levels increase in the zebrafish after 5 days-post-fertilization (dpf), when they start to feed from external sources (Thomason et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, at this developmental stage the larvae increase sensitivity to Mn exposure (Altenhofen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bakthavatsalam et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nevertheless, none of these studies analyzed the cellular effects of Mn during zebrafish early developmental stages. Although zebrafish get more sensitive after 5 dpf, we have used early stages until 96hpf larvae to investigate in more detail the complexity of larvae phenotypes exposed to Mn. We found that at concentrations of 0.5 mg/L, 5 times above the acceptable for potable water in Brazil (Conama, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), detected in polluted environments (da Silva, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Francisco et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Queiroz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the zebrafish embryos presented a non-inflated swim bladder. Moreover, we describe interesting phenotypes besides the neurological effects in the literature, such as the formation of skin aggregates, cellular infiltration and increased number of melanocytes. Therefore, we aimed to analyze the cellular effects of Mn during zebrafish development using high quality light microscopy and confocal fluorescence microscopy. Mn induced an increased expression of the signaling protein β-catenin on cells beneath the skin aggregates with mesenchymal morphology. Our data suggest a disruptive effect of Mn on skin cellular organization and the regulation of Wnt/β-catenin on mesenchymal cells that were not previously described in the literature.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Zebrafish husbandry and embryo exposure to Mn\u003c/h2\u003e \u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) were maintained in aquaria with recirculating water system at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C on a 14:10 light/dark cycle in animal facility at the Institute of Biodiversity and Sustainability (NUPEM) of the Federal University of Rio de Janeiro, Maca\u0026eacute;-RJ, Brazil according to (Westerfield, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Animals were handled and experimented according to Institutional Animal Care and Use Committee protocols under the number 067/17. Embryos and larvae were collected and treated at an early stage with increasing concentrations of MnCl2 (0.5 mg/L, 5 mg/L, 50 mg/L, 100 mg/L and 500 mg/L) up to 96 hours post fertilization (hpf).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sudan Black labeling\u003c/h2\u003e \u003cp\u003eSudan Black labeling protocol was modified from previous studies (Le Guyader et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sheehan \u0026amp; Storey, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1947\u003c/span\u003e; Walters et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Control and Mn-treated zebrafish larvae at 96-hpf stages were fixed in 4% paraformaldehyde overnight. Next, larvae were washed five times with PBS and incubated with 0.3% Sudan Black Solution (dissolved in ethanol, phenol and Na2HPO.12H2O) for 20 min. Then, larvae were washed five times with 70% ethanol and incubated for 15 min with 1% KOH, 1%H2O2 and EtOH 70%. Larvae were incubated for 10 min with PBS containing 0.1% Tween 20 and 50% ethanol, followed by 10 min with 30% ethanol and digital images of larvae were obtained in a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Antibodies and probes\u003c/h2\u003e \u003cp\u003eRabbit polyclonal antibody against beta-catenin (code # C-2206) was from Sigma-Aldrich (USA). DNA-binding probe 4,6-Diamino-2-phenylindole dihydrochloride (DAPI) and Alexa Fluor 546-goat anti-rabbit IgG antibody was from Molecular Probes (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescence\u003c/h2\u003e \u003cp\u003eDechorionated zebrafish larvae at 96 hpf were fixed in 4% paraformaldehyde in PBS for 1h at room temperature. Larvae were then permeabilized with 0.5% Triton-X 100 in PBS (PBS/T) three times for 30 min and incubated overnight at 4\u0026deg;C with primary antibodies (all diluted 1:100 in PBS/T). Then, larvae were washed for 30 min with PBS/T and incubated for 1 hour at 37\u0026ordm; C with Alexa Fluor-conjugated secondary antibodies (all diluted 1:200 in PBS/T). Nuclei were labeled with 0.1 \u0026micro;g/mL of DAPI in 0.9% NaCl. Larvae were mounted on #1.5 24 x 60-mm glass coverslips (with spacers) using Prolong Gold (Molecular Probes). Experiments with zebrafish larvae were repeated four times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fluorescence image acquisition and processing\u003c/h2\u003e \u003cp\u003eZebrafish larvae were examined in an Axiovert 100 microscope (Carl Zeiss, Germany) coupled to an Olympus DP71 high-resolution camera, and in a DSU Spinning Disk confocal scanner mounted on an inverted fluorescent microscope (Olympus, Japan) and TCS SPE confocal (Leica). For live imaging, live embryos were mounted in 1% methyl cellulose and bright-field microscopy was performed on a Leica stereomicroscope (Leica, Germany). Image processing (brightness, contrast adjustments and area quantification) was performed using Fiji software (Schindelin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and figure panels were mounted with Adobe Photoshop software (Adobe Systems Inc., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out using the GraphPad Prism software version 8. The results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The groups were performed with at least three independent experiments (N\u0026thinsp;=\u0026thinsp;3), where ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. control group. Statistical analysis was performed using one-way ANOVA.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Analysis of mortality, hatching and alterations in swim bladder and heart after in zebrafish larvae after manganese treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the effects of manganese (Mn) in zebrafish embryos, we first analyzed the mortality rate (\u003cstrong\u003eSupplemental Fig.\u0026nbsp;1\u003c/strong\u003e) of zebrafish larvae exposed to different concentrations of Mn (0.5, 5, 50, 100 and 500 mg/L). The mortality rate for all concentrations until 96 hpf were below 10% (\u003cstrong\u003eSupplemental Fig.\u0026nbsp;1\u003c/strong\u003e). It is comparable to what has been described in the literature, where the mortality to Mn exposure increase after 96 hpf (Altenhofen et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bakthavatsalam et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tu et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). However hatching rate at 500 mg/L was significantly impaired with strong morphological retardation of the embryos at 96 hpf (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). It is possible that non-hatching embryos exposed to 500 mg/L (9 mM) would not survive after 5dpf. If we consider the LC50 of embryo-larvae exposed to MnCl2 for 48h after 3.5 dpf the LC50 was of 2.0 mM (Bakthavatsalam et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). In another study, the embryos exposed from 48 hpf-120 hpf had a LC50 range of 6.0-10mM (Hern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eRegarding the morphological phenotypes, apart from neurological and craniofacial morphological malformations observed in the literature (Altenhofen et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bakthavatsalam et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tu et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), we found alterations in swim bladder, heart and cell aggregates on the skin of zebrafish larvae. Swimbladder fails to inflate in 60% of the larvae in 0.5 mg/L and 77% in 5 mg/L (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). While all embryos exposed to 50, 100 and 500 mg/L of Mn presented influence in swim bladder inflation (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-I). The Mn concentration exposure started with 0.5 mg/L that is similar to what has been found in polluted environmental samples (da Silva, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Francisco et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Queiroz et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) and is above the 0.1 mg/L permitted in potable water in Brazil (Conama, \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). The swim bladder inflates at 4.5 dpf when the larvae have 4.4mm (Lindsey et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Winata et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is a gas-filled organ that is important for fish buoyancy and has similarities to tetrapod lungs (Lindsey et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Winata et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). This data reinforces the importance of keeping the Mn levels in the water below 0.5 mg/L.\u003c/p\u003e\n\u003cp\u003eAnother phenotype noticed in zebrafish embryos was cardiac edema, which was detected in 30% of the larvae in 50 mg/L, 50% in 100 mg/L and 25% in 500 mg/L (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). Indeed, Mn has been shown to cause heart disturbance in many vertebrates including humans (Cr\u0026eacute;ton et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Jiang \u0026amp; Zheng, \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). Workers exposed to Mn present higher alterations in electrocardiogram (ECG) than control group, caused by different types of cardiac alterations (Jiang \u0026amp; Zheng, \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). In zebrafish embryos, Mn induce calcium decrease (Hern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) and pericardial edema (Tu et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides calcium dysregulation leads to failure of heart development in zebrafish (Cr\u0026eacute;ton et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e), calcium itself has important roles along zebrafish development (Webb \u0026amp; Miller, \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). In case calcium signaling disruption in the embryo would be proportional to Mn concentration, it could mean that as higher the concentration of Mn, more calcium imbalance would be generated. This could be one of the explanations why the embryos exposed to 500 mg/L had a lower percentage of heart edema than the ones exposed to 100 mg/L, including its strong impairment in development (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). However, this should be further investigated in future work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Manganese treatment induces an increase in melanocyte area and the formation of skin aggregates in zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLarvae treated with 500 mg/L Mn had severe development impairment, therefore, we carried out the detailed phenotype investigation of 50 and 100mg/L Mn treated embryos. At 96hpf, the larvae had a significant decrease in the total length after 100 mg/L of Mn (6% decrease compared to untreated larvae) (\u003cstrong\u003eSupplemental Fig.\u0026nbsp;2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFurther analysis of zebrafish larvae caudal fin size under the light microscope, exhibited an extended area of melanocyte occupation after Mn exposure. Quantification of the melanocyte area showed an 50% increase after 50 mg/L Mn, whereas no change was observed after 100 mg/L Mn (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, Mn has a high affinity for melanin tissues \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Lyd\u0026eacute;n et al., \u003cspan class=\"CitationRef\"\u003e1984\u003c/span\u003e) and accumulate in specific regions of the brain, especially in pigmented neurons of \u003cem\u003esubstantia nigra\u003c/em\u003e (O\u0026rsquo;Neal \u0026amp; Zheng, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The toxicity of Mn in the nervous system has been widely described, but little data is related to skin melanocytes. There is a poster abstract reporting that human melanocytes viability decreases in a dose dependent manner upon Mn exposure (Kim et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). It remains to be investigated the response of melanocytes to Mn, related to growth and lethality. Mn in the brain induce mitochondrial impairment that lead to a series of events ending in apoptosis induction (reviewed in Pajarillo et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Would it be the same for other melanin-containing cells?\u003c/p\u003e\n\u003cp\u003eWe analyzed another group of cells that presented abnormal behavior that were not previously described in the literature. One of the unexpected phenotypes found was the presence of skin aggregates on the larvae. We found a 1.5 increase in skin aggregates in the larvae treated with 50 mg/L Mn (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The cellular aggregates were clearly visible under the light microscope without any kind of labeling or staining. Quantification of the size of the cellular aggregates showed that they have an average size of 1.53 mm\u003csup\u003e2\u003c/sup\u003e after 50 mg/L of Mn and 0.43 mm\u003csup\u003e2\u003c/sup\u003e in 100 mg/L Mn (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Previous reports of Mn toxicity in zebrafish larvae did not cite those skin aggregates. The tail fin is a very fragile structure. Possibly the larvae preparation used for analyses, smashed the tail fins and this phenotype was overlooked (Bakthavatsalam et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tu et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is known that Mn deficient diets in young men lead to skin rashes (Friedman et al., \u003cspan class=\"CitationRef\"\u003e1987\u003c/span\u003e). On the other hand, patients exposed to MnCl\u003csub\u003e2\u003c/sub\u003e presented irritant reactions and it was cytotoxic to keratinocytes cell culture (Shallcross et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting an important role of Mn in skin homeostasis. Ionic imbalance in zebrafish \u003cem\u003eatpase1b1a\u003c/em\u003e mutant, a mutation in Na,K-ATPase \u0026beta;1-subunit, demonstrated the formation of skin aggregates derived from the proliferation of basal keratinocytes with metastasis capacity (Hatzold et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The skin aggregates after Mn exposure were morphologically very similar to \u003cem\u003eatpase1b1a\u003c/em\u003e mutant. Mn affect the expression and/or function of other atpases as ATP13A2 and ATP2C1 and calcium balance (Gavin et al., \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e; Tan et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Xia et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The aggregates from \u003cem\u003eatpase1b1a\u003c/em\u003e mutants produced metalloproteinase-9 (MMP) capable of degrade basement membrane and is a malignancy marker (Hatzold et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Mn induces MMP9 expression in human keratinocytes (Chebassier et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e) and rat astrocytes (Latronico et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Future experiments should characterize if the skin aggregates have similar malignant characteristics as the ones formed by ionic disturbances.\u003c/p\u003e\n\u003cp\u003eAnother important characteristic of Mn toxicity is the induction of an inflammatory environment, inducing interleukin-6 and tumor necrosis factor \u0026alpha; (Kobayashi et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Milatovic et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shallcross et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). To further analyze for cell infiltrates possibly induced by inflammation, we have stained 96 hpf larvae with Sudan Black (SB) (Le Guyader et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sheehan \u0026amp; Storey, \u003cspan class=\"CitationRef\"\u003e1947\u003c/span\u003e). SB stain rich lipid organelles of granulocytes and has been characterized in zebrafish to stain myeloperoxidase-positive granules of neutrophils (Le Guyader et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). SB stained the cellular infiltrates in the caudal fin of zebrafish larvae, and the area of aggregates had SB-positive cells after Mn treatment, as compared to untreated larvae (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating an inflammatory response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Manganese treatment induces an accumulation of beta-catenin in aggregates in the caudal fin of zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe disturbances in cell organization in the caudal fin of zebrafish larvae after Mn treatment raise the idea to analyze the distribution of proteins related to tissue structure. \u0026beta;-catenin is a structural and signaling protein that is essential for the generation and maintenance of epithelial tissues organization (van der Wal \u0026amp; van Amerongen, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u0026beta;-catenin localization in the nucleus indicates activation of the canonical Wnt/\u0026beta;-catenin pathway (van der Wal \u0026amp; van Amerongen, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Immunofluorescence labeling of \u0026beta;-catenin and the nuclear dye DAPI showed \u0026beta;-catenin in 96 hpf larvae control were more diffuse along the skin cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). However, in Mn exposed embryos, \u0026beta;-catenin was concentrated in cells beneath the caudal fin aggregates; and not in the cell aggregates (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-I). In higher magnification, it is possible to see that \u0026beta;-catenin-positive cells have an elongated morphology, resembling mesenchymal migrating cells below the skin (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). However, the staining is not all over the dermal-mesenchymal cells that migrate into the caudal fins (Feitosa et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is possible that they could be myeloid migrating cells. Further investigation on the nature of these positive-\u0026beta;-catenin cells should be analyzed. However it is exciting to think that there is a cross-talk between NF\u0026kappa;B and Wnt/\u0026beta;-catenin in inflammatory events, acting positively or negatively (Ma \u0026amp; Hottiger, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Recent literature demonstrates that ficetin, a flavonoid, activated \u0026beta;-catenin in zebrafish larvae and induced melanogenesis (Molagoda et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) and in macrophages the \u0026beta;-catenin activation can inhibit the NF\u0026kappa;B signaling, acting as an anti-inflammatory (Molagoda et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). This leads to future questions to be answered on the role of Mn toxicity in \u0026beta;-catenin activation in mesenchymal cells with the potential to act as an inflammatory mediator in skin damage.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eHere, we studied the effects of manganese (Mn) during zebrafish development and found that concentration of 50 and 100 mg/L of Mn (i) induces alterations in swim bladder inflation, heart, and size of zebrafish larvae, (ii) induces an increase in melanocyte area and the formation of cellular aggregates in the caudal fin, and (iii) induces an accumulation of β-catenin in mesenchymal cells in the caudal fin of zebrafish larvae. These results open important new questions on the Mn role in skin cell organization, melanocyte behavior and the multifunctional protein β-catenin in the cellular responses in zebrafish.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated for this study can be found within the manuscript figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of zebrafish embryos was approved by the Ethics Committee for Animal Care and Use in Scientific Research from the Federal University of Rio de Janeiro and received the approval number 067/17.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions- Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMF conceived the work. GG and JLO contributed to the acquisition of data. CM, GG, JLO, MC and NMF contributed to the analysis and interpretation of data. CM and NF wrote the first draft of the manuscript. All authors consent to participate, contributed to manuscript revision, read, and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll listed authors consent and agree with the publication of the data present within this manuscript. The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, funding number 302115/2017-0 for CM, 301443/2018-1 for MLC, 431352/2018-6 for NMF) and Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, funding number E-26/202.920/2019 for CM and E26/210.220/2018 for MLC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAltenhofen S, Wiprich MT, Nery LR, Leite CE, Vianna MR, M. 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Here we studied the effects of Mn in zebrafish, at the cellular level, due to the transparent nature of zebrafish larvae that enables a powerful analysis under the light microscope. The collection of our results shows that environmental concentrations of 0.5 mg/L affect swimbladder inflation; at concentration of 50 mg/L and 100 mg/L Mn (i) induces alterations in viability, swim bladder, heart, and size of zebrafish larvae, (ii) induces an increase in melanocyte area and the formation of cellular aggregates in the skin, and (iii) induces an accumulation of β-catenin in mesenchymal cells in the caudal fin of zebrafish larvae. Our data suggest that increased levels of Mn induce cell aggregate formation in the skin and the presence of more melanocytes in the zebrafish caudal fin. Interestingly, the adhesion protein β-catenin was activated in mesenchymal cells near the cell aggregates. These results open important new questions on the role of Mn toxicity on cellular organization and β-catenin responses in fishes.\u003c/p\u003e","manuscriptTitle":"Manganese exposure induces cellular aggregates and the accumulation of β-catenin in skin of zebrafish embryos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-31 13:57:50","doi":"10.21203/rs.3.rs-1570773/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-05-19T03:30:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-18T18:24:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2022-05-09T13:22:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-06T05:32:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2022-04-18T22:35:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71d5af14-b795-4bd9-babd-fc2c7ac40207","owner":[],"postedDate":"May 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-05-31T13:57:50+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-31 13:57:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1570773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1570773","identity":"rs-1570773","version":["v1"]},"buildId":"qQ7_6M8ijIrYJ9CiyUnPg","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}