Danio rerio; zebrafish; transgenic lines; bone matrix; probiotics; mineralization; BMP inhibitors; bone growth
Abstract :
[en] Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854).
Research center :
GIGA-I3 - Giga-Infection, Immunity and Inflammation - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Sojan, Jerry Maria ✱; Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy > Department of Life and Environmental Sciences
Raman, Ratish ✱; Université de Liège - ULiège > GIGA > GIGA I3 - Laboratory for Organogenesis and Regeneration
Muller, Marc ; Université de Liège - ULiège > GIGA > GIGA I3 - Laboratory for Organogenesis and Regeneration
Carnevali, Oliana
Renn, Jörg ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
✱ These authors have contributed equally to this work.
Language :
English
Title :
Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health
Publication date :
29 April 2022
Journal title :
International Journal of Molecular Sciences
ISSN :
1661-6596
eISSN :
1422-0067
Publisher :
Multidisciplinary Digital Publishing Institute (MDPI), Switzerland
Hill, C.; Guarner, F.; Reid, G.; Gibson, G.R.; Merenstein, D.J.; Pot, B.; Morelli, L.; Canani, R.B.; Flint, H.J.; Salminen, S.; et al. The International Scientific Association for Probiotics and Prebiotics Consensus Statement on the Scope and Appropriate Use of the Term Probiotic. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 506–514. [CrossRef] [PubMed]
McCabe, L.; Britton, R.A.; Parameswaran, N. Prebiotic and Probiotic Regulation of Bone Health: Role of the Intestine and Its Microbiome. Curr. Osteoporos. Rep. 2015, 13, 363–371. [CrossRef]
Ohlsson, C.; Sjögren, K. Osteomicrobiology: A New Cross-Disciplinary Research Field. Calcif. Tissue Int. 2018, 102, 426–432. [CrossRef] [PubMed]
Rizzoli, R.; Biver, E. Are Probiotics the New Calcium and Vitamin D for Bone Health? Curr. Osteoporos. Rep. 2020, 18, 273–284. [CrossRef] [PubMed]
Cosme-Silva, L.; Dal-Fabbro, R.; Cintra, L.T.A.; Ervolino, E.; Plazza, F.; Mogami Bomfim, S.; Duarte, P.C.T.; Junior, V.E.D.S.; Gomes-Filho, J.E. Reduced Bone Resorption and Inflammation in Apical Periodontitis Evoked by Dietary Supplementation with Probiotics in Rats. Int. Endod. J. 2020, 53, 1084–1092. [CrossRef] [PubMed]
Gholami, A.; Dabbaghmanesh, M.H.; Ghasemi, Y.; Talezadeh, P.; Koohpeyma, F.; Montazeri-Najafabady, N. Probiotics Ameliorate Pioglitazone-Associated Bone Loss in Diabetic Rats. Diabetol. Metab. Syndr. 2020, 12, 78. [CrossRef]
Huidrom, S.; Beg, M.A.; Masood, T. Post-Menopausal Osteoporosis and Probiotics. Curr. Drug Targets 2021, 22, 816–822. [CrossRef] [PubMed]
Jia, L.; Tu, Y.; Jia, X.; Du, Q.; Zheng, X.; Yuan, Q.; Zheng, L.; Zhou, X.; Xu, X. Probiotics Ameliorate Alveolar Bone Loss by Regulating Gut Microbiota. Cell Prolif. 2021, 54, e13075. [CrossRef]
Britton, R.A.; Irwin, R.; Quach, D.; Schaefer, L.; Zhang, J.; Lee, T.; Parameswaran, N.; McCabe, L.R.; Probiotic, L. Reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model. J. Cell. Physiol. 2014, 229, 1822–1830. [CrossRef]
Chiang, S.-S.; Pan, T.-M. Antiosteoporotic Effects of Lactobacillus-Fermented Soy Skim Milk on Bone Mineral Density and the Microstructure of Femoral Bone in Ovariectomized Mice. J. Agric. Food Chem. 2011, 59, 7734–7742. [CrossRef]
Ohlsson, C.; Engdahl, C.; Fåk, F.; Andersson, A.; Windahl, S.H.; Farman, H.H.; Movérare-Skrtic, S.; Islander, U.; Sjögren, K. Probiotics Protect Mice from Ovariectomy-Induced Cortical Bone Loss. PLoS ONE 2014, 9, e92368. [CrossRef]
Parvaneh, K.; Ebrahimi, M.; Sabran, M.R.; Karimi, G.; Hwei, A.N.M.; Abdul-Majeed, S.; Ahmad, Z.; Ibrahim, Z.; Jamaluddin, R. Probiotics (Bifidobacterium Longum) Increase Bone Mass Density and Upregulate Sparc and Bmp-2 Genes in Rats with Bone Loss Resulting from Ovariectomy. BioMed. Res. Int. 2015, 2015, e897639. [CrossRef]
Foureaux, R.d.C.; Messora, M.R.; de Oliveira, L.F.F.; Napimoga, M.H.; Pereira, A.N.J.; Ferreira, M.S.; Pereira, L.J. Effects of Probiotic Therapy on Metabolic and Inflammatory Parameters of Rats with Ligature-Induced Periodontitis Associated with Restraint Stress. J. Periodontol. 2014, 85, 975–983. [CrossRef] [PubMed]
Nilsson, A.G.; Sundh, D.; Bäckhed, F.; Lorentzon, M. Lactobacillus Reuteri Reduces Bone Loss in Older Women with Low Bone Mineral Density: A Randomized, Placebo-Controlled, Double-Blind, Clinical Trial. J. Intern. Med. 2018, 284, 307–317. [CrossRef] [PubMed]
Takimoto, T.; Hatanaka, M.; Hoshino, T.; Takara, T.; Tanaka, K.; Shimizu, A.; Morita, H.; Nakamura, T. Effect of Bacillus Subtilis C-3102 on Bone Mineral Density in Healthy Postmenopausal Japanese Women: A Randomized, Placebo-Controlled, Double-Blind Clinical Trial. Biosci. Microbiota Food Health 2018, 37, 87–96. [CrossRef] [PubMed]
Maradonna, F.; Gioacchini, G.; Falcinelli, S.; Bertotto, D.; Radaelli, G.; Olivotto, I.; Carnevali, O. Probiotic Supplementation Promotes Calcification in Danio Rerio Larvae: A Molecular Study. PLoS ONE 2013, 8, e83155. [CrossRef]
Terashima, A.; Takayanagi, H. Overview of Osteoimmunology. Calcif. Tissue Int. 2018, 102, 503–511. [CrossRef] [PubMed]
Wu, S.; Yoon, S.; Zhang, Y.-G.; Lu, R.; Xia, Y.; Wan, J.; Petrof, E.O.; Claud, E.C.; Chen, D.; Sun, J. Vitamin D Receptor Pathway Is Required for Probiotic Protection in Colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 2015, 309, G341–G349. [CrossRef]
Li, J.-Y.; Chassaing, B.; Tyagi, A.M.; Vaccaro, C.; Luo, T.; Adams, J.; Darby, T.M.; Weitzmann, M.N.; Mulle, J.G.; Gewirtz, A.T.; et al. Sex Steroid Deficiency–Associated Bone Loss Is Microbiota Dependent and Prevented by Probiotics. J. Clin. Investig. 2016, 126, 2049–2063. [CrossRef]
Chaplin, A.; Parra, P.; Laraichi, S.; Serra, F.; Palou, A. Calcium Supplementation Modulates Gut Microbiota in a Prebiotic Manner in Dietary Obese Mice. Mol. Nutr. Food Res. 2016, 60, 468–480. [CrossRef]
Atkins, G.J.; Welldon, K.J.; Wijenayaka, A.R.; Bonewald, L.F.; Findlay, D.M. Vitamin K Promotes Mineralization, Osteoblast-to-Osteocyte Transition, and an Anticatabolic Phenotype by γ-Carboxylation-Dependent and-Independent Mechanisms. Am. J. Physiol. Cell Physiol. 2009, 297, C1358–C1367. [CrossRef] [PubMed]
Booth, S.L. Roles for Vitamin K Beyond Coagulation. Annu. Rev. Nutr. 2009, 29, 89–110. [CrossRef] [PubMed]
Castaneda, M.; Strong, J.M.; Alabi, D.A.; Hernandez, C.J. The Gut Microbiome and Bone Strength. Curr. Osteoporos. Rep. 2020, 18, 677–683. [CrossRef] [PubMed]
Lleras-Forero, L.; Winkler, C.; Schulte-Merker, S. Zebrafish and Medaka as Models for Biomedical Research of Bone Diseases. Dev. Biol. 2020, 457, 191–205. [CrossRef] [PubMed]
Bergen, D.J.M.; Kague, E.; Hammond, C.L. Zebrafish as an Emerging Model for Osteoporosis: A Primary Testing Platform for Screening New Osteo-Active Compounds. Front. Endocrinol. 2019, 10, 6. [CrossRef] [PubMed]
Sun, X.; Zhang, R.; Chen, H.; Du, X.; Chen, S.; Huang, J.; Liu, M.; Xu, M.; Luo, F.; Jin, M.; et al. Fgfr3 Mutation Disrupts Chondrogenesis and Bone Ossification in Zebrafish Model Mimicking CATSHL Syndrome Partially via Enhanced Wnt/β-Catenin Signaling. Theranostics 2020, 10, 7111–7130. [CrossRef]
Jacobs, C.T.; Huang, P. Notch Signalling Maintains Hedgehog Responsiveness via a Gli-Dependent Mechanism during Spinal Cord Patterning in Zebrafish. eLife 2019, 8, e49252. [CrossRef]
Alhazmi, N.; Carroll, S.H.; Kawasaki, K.; Woronowicz, K.C.; Hallett, S.A.; Macias Trevino, C.; Li, E.B.; Baron, R.; Gori, F.; Yelick, P.C.; et al. Synergistic Roles of Wnt Modulators R-Spondin2 and R-Spondin3 in Craniofacial Morphogenesis and Dental Development. Sci. Rep. 2021, 11, 5871. [CrossRef]
Lovely, C.B.; Swartz, M.E.; McCarthy, N.; Norrie, J.L.; Eberhart, J.K. Bmp Signaling Mediates Endoderm Pouch Morphogenesis by Regulating Fgf Signaling in Zebrafish. Development 2016, 143, 2000–2011. [CrossRef]
Schiavone, M.; Rampazzo, E.; Casari, A.; Battilana, G.; Persano, L.; Moro, E.; Liu, S.; Leach, S.D.; Tiso, N.; Argenton, F. Zebrafish Reporter Lines Reveal in Vivo Signaling Pathway Activities Involved in Pancreatic Cancer. Dis. Model Mech. 2014, 7, 883–894. [CrossRef]
Westphal, M.; Panza, P.; Kastenhuber, E.; Wehrle, J.; Driever, W. Wnt/β-Catenin Signaling Promotes Neurogenesis in the Diencephalospinal Dopaminergic System of Embryonic Zebrafish. Sci. Rep. 2022, 12, 1030. [CrossRef] [PubMed]
Ando, K.; Shibata, E.; Hans, S.; Brand, M.; Kawakami, A. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance. Dev. Cell 2017, 43, 643–650.e3. [CrossRef]
Cooney, O.D.; Nagareddy, P.R.; Murphy, A.J.; Lee, M.K.S. Healthy Gut, Healthy Bones: Targeting the Gut Microbiome to Promote Bone Health. Front. Endocrinol. 2021, 11, 1159. [CrossRef] [PubMed]
Renn, J.; Pruvot, B.; Muller, M. Detection of Nitric Oxide by Diaminofluorescein Visualizes the Skeleton in Living Zebrafish. J. Appl. Ichthyol. 2014, 30, 701–706. [CrossRef]
Debiais-Thibaud, M.; Simion, P.; Ventéo, S.; Muñoz, D.; Marcellini, S.; Mazan, S.; Haitina, T. Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates. Mol. Biol. Evol. 2019, 36, 2265–2276. [CrossRef]
Huitema, L.F.A.; Apschner, A.; Logister, I.; Spoorendonk, K.M.; Bussmann, J.; Hammond, C.L.; Schulte-Merker, S. Entpd5 Is Essential for Skeletal Mineralization and Regulates Phosphate Homeostasis in Zebrafish. Proc. Natl. Acad. Sci. USA 2012, 109, 21372–21377. [CrossRef]
Zinck, N.W.; Jeradi, S.; Franz-Odendaal, T.A. Elucidating the Early Signaling Cues Involved in Zebrafish Chondrogenesis and Cartilage Morphology. J. Exp. Zool. B Mol. Dev. Evol. 2021, 336, 18–31. [CrossRef]
Windhausen, T.; Squifflet, S.; Renn, J.; Muller, M. BMP Signaling Regulates Bone Morphogenesis in Zebrafish through Promoting Osteoblast Function as Assessed by Their Nitric Oxide Production. Molecules 2015, 20, 7586–7601. [CrossRef]
Azetsu, Y.; Inohaya, K.; Takano, Y.; Kinoshita, M.; Tasaki, M.; Kudo, A. The Sp7 Gene Is Required for Maturation of Osteoblast-Lineage Cells in Medaka (Oryzias Latipes) Vertebral Column Development. Dev. Biol. 2017, 431, 252–262. [CrossRef]
Hammond, C.L.; Moro, E. Using Transgenic Reporters to Visualize Bone and Cartilage Signaling during Development in Vivo. Front. Endocrinol. 2012, 3, 91. [CrossRef] [PubMed]
Spoorendonk, K.M.; Peterson-Maduro, J.; Renn, J.; Trowe, T.; Kranenbarg, S.; Winkler, C.; Schulte-Merker, S. Retinoic Acid and Cyp26b1 Are Critical Regulators of Osteogenesis in the Axial Skeleton. Development 2008, 135, 3765–3774. [CrossRef] [PubMed]
Singh, S.P.; Holdway, J.E.; Poss, K.D. Regeneration of Amputated Zebrafish Fin Rays from de Novo Osteoblasts. Dev. Cell 2012, 22, 879–886. [CrossRef] [PubMed]
Renn, J.; Winkler, C. Osterix-MCherry Transgenic Medaka for in Vivo Imaging of Bone Formation. Dev. Dyn. 2009, 238, 241–248. [CrossRef]
Wiweger, M.I.; Zhao, Z.; van Merkesteyn, R.J.P.; Roehl, H.H.; Hogendoorn, P.C.W. HSPG-Deficient Zebrafish Uncovers Dental Aspect of Multiple Osteochondromas. PLoS ONE 2012, 7, e29734. [CrossRef]
Clément, A.; Wiweger, M.; von der Hardt, S.; Rusch, M.A.; Selleck, S.B.; Chien, C.-B.; Roehl, H.H. Regulation of Zebrafish Skeletogenesis by Ext2/Dackel and Papst1/Pinscher. PLoS Genet. 2008, 4, e1000136. [CrossRef]
Yan, Y.-L.; Bhattacharya, P.; He, X.J.; Ponugoti, B.; Marquardt, B.; Layman, J.; Grunloh, M.; Postlethwait, J.H.; Rubin, D.A. Duplicated Zebrafish Co-Orthologs of Parathyroid Hormone-Related Peptide (PTHrP, Pthlh) Play Different Roles in Craniofacial Skeletogenesis. J. Endocrinol. 2012, 214, 421–435. [CrossRef]
Reijntjes, S.; Rodaway, A.; Maden, M. The Retinoic Acid Metabolising Gene, CYP26B1, Patterns the Cartilaginous Cranial Neural Crest in Zebrafish. Int. J. Dev. Biol. 2003, 51, 351–360. [CrossRef]
Laue, K.; Jänicke, M.; Plaster, N.; Sonntag, C.; Hammerschmidt, M. Restriction of Retinoic Acid Activity by Cyp26b1 Is Required for Proper Timing and Patterning of Osteogenesis during Zebrafish Development. Development 2008, 135, 3775–3787. [CrossRef]
Laue, K.; Pogoda, H.-M.; Daniel, P.B.; van Haeringen, A.; Alanay, Y.; von Ameln, S.; Rachwalski, M.; Morgan, T.; Gray, M.J.; Breuning, M.H.; et al. Craniosynostosis and Multiple Skeletal Anomalies in Humans and Zebrafish Result from a Defect in the Localized Degradation of Retinoic Acid. Am. J. Hum. Genet. 2011, 89, 595–606. [CrossRef]
Maruyama, Z.; Yoshida, C.A.; Furuichi, T.; Amizuka, N.; Ito, M.; Fukuyama, R.; Miyazaki, T.; Kitaura, H.; Nakamura, K.; Fujita, T.; et al. Runx2 Determines Bone Maturity and Turnover Rate in Postnatal Bone Development and Is Involved in Bone Loss in Estrogen Deficiency. Dev. Dyn. 2007, 236, 1876–1890. [CrossRef] [PubMed]
Komori, T. Regulation of Bone Development and Extracellular Matrix Protein Genes by RUNX2. Cell Tissue Res. 2009, 339, 189. [CrossRef]
Lowery, J.W.; Rosen, V. The BMP Pathway and Its Inhibitors in the Skeleton. Physiol. Rev. 2018, 98, 2431–2452. [CrossRef] [PubMed]
Kimura, Y.; Hisano, Y.; Kawahara, A.; Higashijima, S. Efficient Generation of Knock-in Transgenic Zebrafish Carrying Re-porter/Driver Genes by CRISPR/Cas9-Mediated Genome Engineering. Sci. Rep. 2014, 4, 6545. [CrossRef]
Dalcq, J.; Pasque, V.; Ghaye, A.; Larbuisson, A.; Motte, P.; Martial, J.A.; Muller, M. RUNX3, EGR1 and SOX9B Form a Regulatory Cascade Required to Modulate BMP-Signaling during Cranial Cartilage Development in Zebrafish. PLoS ONE 2012, 7, e50140. [CrossRef] [PubMed]
Larbuisson, A.; Dalcq, J.; Martial, J.A.; Muller, M. Fgf Receptors Fgfr1a and Fgfr2 Control the Function of Pharyngeal Endoderm in Late Cranial Cartilage Development. Differentiation 2013, 86, 192–206. [CrossRef]
Maradonna, F.; Nozzi, V.; Dalla Valle, L.; Traversi, I.; Gioacchini, G.; Benato, F.; Colletti, E.; Gallo, P.; Di Marco Pisciottano, I.; Mita, D.G.; et al. A Developmental Hepatotoxicity Study of Dietary Bisphenol A in Sparus Aurata Juveniles. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 2014, 166, 1–13. [CrossRef]
Carnevali, O.; Notarstefano, V.; Olivotto, I.; Graziano, M.; Gallo, P.; Di Marco Pisciottano, I.; Vaccari, L.; Mandich, A.; Giorgini, E.; Maradonna, F. Dietary Administration of EDC Mixtures: A Focus on Fish Lipid Metabolism. Aquat. Toxicol. 2017, 185, 95–104. [CrossRef]
Bensimon-Brito, A.; Cardeira, J.; Dionísio, G.; Huysseune, A.; Cancela, M.L.; Witten, P.E. Revisiting in Vivo Staining with Alizarin Red S—A Valuable Approach to Analyse Zebrafish Skeletal Mineralization during Development and Regeneration. BMC Dev. Biol. 2016, 16, 2. [CrossRef]
R Core Team, R. A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2020. Available online: https://www.yumpu.com/s/Fxit5xIdza5bKZeV (accessed on 24 March 2022).