Articles
Vol. 69 No. 4 (2025)
https://doi.org/10.4081/ejh.2025.4242

Expression of nestin, parvalbumin and otoferlin during cochlear development in the mouse: an immunofluorescence study

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Published: 22 September 2025
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Data Availability Statement

the datasets used in the current study are available upon reasonable request from the corresponding author.

Nestin, parvalbumin, otoferlin, immunofluorescence, mouse, cochlea

Authors

Wenjing Liu
Otolaryngology & Head and Neck Center, Cancer Center, Department of Otolaryngology, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College), Hangzhou, China.
Yongchun Zhang
Department of Otorhinolaryngology-Head and Neck Surgery, Zhongda Hospital, Southeast University, Nanjing, China.
Cheng Liang
Department of Otorhinolaryngology-Head and Neck Surgery, Zhongda Hospital, Southeast University, Nanjing, China.
Shujuan Yang
ysjsrmyy@163.com
Otolaryngology & Head and Neck Center, Cancer Center, Department of Otolaryngology, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College), Hangzhou, China.

To elucidate the proteins associated with cochlear development and auditory formation from a histomorphological point of view, this study examined the spatio-temporal expression pattern of nestin, parvalbumin, and otoferlin in the mouse cochlea from embryonic day 17 (E17) to postnatal day 28 (P28) using immunofluorescence. Our findings revealed that nestin was broadly expressed in developing otic mesenchyme cells beneath the basilar membrane, medial to the greater epithelial ridge, and adjacent to the developing stria vascularis during late embryonic stages (E17 and E18.5). From P1 to the onset of hearing (P14), nestin was primarily expressed in fibrocytes derived from otic mesenchyme cells in the spiral ligament and spiral limbus, as well as in tympanic border cells. Dual immunofluorescence staining of nestin with Isolectin B4 (IB4), a specific vascular endothelial marker, showed the location of nestin in the blood vessels within the cochlear lateral wall. Notably, in adults (P28), nestin expression was downregulated in the fibrocytes of the spiral ligament and spiral limbus but persisted in the tympanic border cells. Parvalbumin immunolabeling was consistently observed in spiral ganglion neurons (SGNs) and inner hair cells (IHCs) from E17 through adulthood. By P1, parvalbumin expression extended to all three rows of outer hair cells (OHCs) and persisted into adulthood. Transient parvalbumin expression was also noted in afferent nerve fibers innervating the IHCs during early postnatal stages. Otoferlin labeling was predominantly detected in the cytoplasm of IHCs, with limited temporal expression in OHCs from P6 to P10. Taken together, these results illustrated the dynamic expression of nestin, parvalbumin and otoferlin during cochlear development and suggested their important function in cochlear development.

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1. Liu W, Chen H, Zhu X, Yu H. Expression of calbindin-D28K in the developing and adult mouse cochlea. J Histochem Cytochem 2022;70:583-96. DOI: https://doi.org/10.1369/00221554221119543
2. Liu W, Zhang Y, Liang C, Jiang X. Developmental expression of calretinin in the mouse cochlea. Eur J Histochem 2024;68:4137. DOI: https://doi.org/10.4081/ejh.2024.4137
3. Liu W, Zhang Y, Liang C, Su L. Expression of S100β during mouse cochlear development. Eur J Histochem 2025;69:4189. DOI: https://doi.org/10.4081/ejh.2025.4189
4. Liu WJ, Ming SS, Zhao XB, Zhu X, Gong YX. Developmental expression of high-mobility group box 1 (HMGB1) in the mouse cochlea. Eur J Histochem 2023;67:3704. DOI: https://doi.org/10.4081/ejh.2023.3704
5. Liu WJ, Yang J. Preferentially regulated expression of connexin 43 in the developing spiral ganglion neurons and afferent terminals in post-natal rat cochlea. Eur J Histochem 2015;59:2464. DOI: https://doi.org/10.4081/ejh.2015.2464
6. Hosoya M, Fujioka M, Murayama AY, Okano H, Ogawa K. The common marmoset as suitable nonhuman alternative for the analysis of primate cochlear development. FEBS J 2021;288:325-53. DOI: https://doi.org/10.1111/febs.15341
7. Nikolic P, Housley GD, Luo L, Ryan AF, Thorne PR. Transient expression of P2X(1) receptor subunits of ATP-gated ion channels in the developing rat cochlea. Brain Res Dev Brain Res 2001;126:173-82. DOI: https://doi.org/10.1016/S0165-3806(00)00149-8
8. Sharlin DS, Visser TJ, Forrest D. Developmental and cell-specific expression of thyroid hormone transporters in the mouse cochlea. Endocrinology 2011;152:5053-64. DOI: https://doi.org/10.1210/en.2011-1372
9. Kiernan AE, Erven A, Voegeling S, Peters J, Nolan P, Hunter J, et al. ENU mutagenesis reveals a highly mutable locus on mouse Chromosome 4 that affects ear morphogenesis. Mamm Genome 2002;13:142-8. DOI: https://doi.org/10.1007/s0033501-2088-9
10. Liu X, Zhao M, Xie Y, Li P, Wang O, Zhou B, et al. Null mutation of the Fascin2 gene by TALEN leading to progressive hearing loss and retinal degeneration in C57BL/6J mice. G3 (Bethesda) 2018;8:3221-30. DOI: https://doi.org/10.1534/g3.118.200405
11. Petitpré C, Faure L, Uhl P, Fontanet P, Filova I, Pavlinkova G, et al. Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification. Nat Commun 2022;13:3878. DOI: https://doi.org/10.1038/s41467-022-31580-1
12. Grandi FC, De Tomasi L, Mustapha M. Single-cell RNA analysis of type i spiral ganglion neurons reveals a Lmx1a population in the cochlea. Front Mol Neurosci 2020;13:83. DOI: https://doi.org/10.3389/fnmol.2020.00083
13. Whitlon DS, Szakaly R, Greiner MA. Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization. Brain Res Protoc 2001;6:159. DOI: https://doi.org/10.1016/S1385-299X(00)00048-9
14. Sharma P, Alsharif S, Fallatah A, Chung BM. Intermediate filaments as effectors of cancer development and metastasis: a focus on keratins, vimentin, and nestin. Cells 2019;8:497-518. DOI: https://doi.org/10.3390/cells8050497
15. Lendahl U, Zimmerman LB, Mckay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585-95. DOI: https://doi.org/10.1016/0092-8674(90)90662-X
16. Selander L, Edlund H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev 2002;113:189-92. DOI: https://doi.org/10.1016/S0925-4773(02)00023-0
17. Lindqvist J, Torvaldson E, Gullmets J, Karvonen H, Nagy A, Taimen P, Eriksson JE. Nestin contributes to skeletal muscle homeostasis and regeneration. J Cell Sci 2017;130:2833-42. DOI: https://doi.org/10.1242/jcs.202226
18. About I, Laurent-Maquin D, Lendahl U, Mitsiadis TA. Nestin expression in embryonic and adult human teeth under normal and pathological conditions. Am J Pathol 2000;157:287-95. DOI: https://doi.org/10.1016/S0002-9440(10)64539-7
19. Lu WJ, Lan F, He Q, Lee A, Tang CZ, Dong L, et al. Inducible expression of stem cell associated intermediate filament nestin reveals an important role in glioblastoma carcinogenesis. Int J Cancer 2011;128:343-51. DOI: https://doi.org/10.1002/ijc.25586
20. Zhang D,Wang J. Nestin is significantly associated with the overall survival of nonsmall cell lung cancer:A meta-analysis. J Cancer Res Ther 2020;16:800-3. DOI: https://doi.org/10.4103/0973-1482.204901
21. Li J, Wang R, Yang L, Wu Q, Wang Q, Nie Z, et al. Knockdown of nestin inhibits proliferation and migration of colorectal cancer cells. Int J Clin Exp Pathol 2015;8:6377-86.
22. Chow CL, Trivedi P, Pyle MP, Matulle JT, Fettiplace R, Gubbels SP. Evaluation of nestin expression in the developing and adult mouse inner ear. Stem Cells Dev 2016;25:1419-32. DOI: https://doi.org/10.1089/scd.2016.0176
23. Oiticica J, Barboza-Junior LC, Batissoco AC, Lezirovitz K, Mingroni-Netto RC, Haddad LA, Bento RF. Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea. J Transl Med 2010;8:119. DOI: https://doi.org/10.1186/1479-5876-8-119
24. Taniguchi M, Yamamoto N, Nakagawa T, Ogino E, Ito J. Identification of tympanic border cells as slow-cycling cells in the cochlea. PLoS One 2012;7:e48544. DOI: https://doi.org/10.1371/journal.pone.0048544
25. Kojima K, Takebayashi S, Nakagawa T, Iwai K, Ito J. Nestin expression in the developing rat cochlea sensory epithelia. Acta Otolaryngol 2004;124:S14-7. DOI: https://doi.org/10.1080/03655230310016744
26. Coppens AG, Salmon I, Heizmann CW, Kiss R, Poncelet L. Postnatal maturation of the dog stria vascularis-- an immunohistochemical study. Anat Rec A Discov Mol Cell Evol Biol 2003;270:82-92. DOI: https://doi.org/10.1002/ar.a.10009
27. Wilwert JL, Madhoun NM, Coughlin DJ. Parvalbumin correlates with relaxation rate in the swimming muscle of sheepshead and kingfish. J Exp Biol 2006;209:227-37. DOI: https://doi.org/10.1242/jeb.01987
28. Bucher EA, Collins JM, King AE, Vickers JC, Kirkcaldie MTK. Coherence and cognition in the cortex: the fundamental role of parvalbumin, myelin, and the perineuronal net. Brain Struct Funct 2021;226:2041-55. DOI: https://doi.org/10.1007/s00429-021-02327-3
29. Rupert DD, Shea SD. Parvalbumin-positive interneurons regulate cortical sensory plasticity in adulthood and development through shared mechanisms. Front Neural Circuits 2022; 16:886629. DOI: https://doi.org/10.3389/fncir.2022.886629
30. Idrizbegovic E, Bogdanovic N, Willott JF, Canlon B. Age-related increases in calcium-binding protein immunoreactivity in the cochlear nucleus of hearing impaired C57BL/6J mice. Neurobiol Aging 2004;25:1085-93. DOI: https://doi.org/10.1016/j.neurobiolaging.2003.11.004
31. Lohmann C, Friauf E. Distribution of the calcium-binding proteins parvalbumin and calretinin in the auditory brainstem of adult and developing rats. J Comp Neurol 1996;367:90-109. DOI: https://doi.org/10.1002/(SICI)1096-9861(19960325)367:1<90::AID-CNE7>3.0.CO;2-E
32. Seto-Ohshima A, Aoki E, Semba R, Emson PC, Heizmann CW. Parvalbumin immunoreactivity in the central auditory system of the gerbil: a developmental study. Neurosci Lett 1990;119:60-3. DOI: https://doi.org/10.1016/0304-3940(90)90755-X
33. Ouda L, Druga R, Syka J. Changes in parvalbumin immunoreactivity with aging in the central auditory system of the rat. Exp Gerontol 2008;43:782-9. DOI: https://doi.org/10.1016/j.exger.2008.04.001
34. Hackney CM, Mahendrasingam S, Penn A, Fettiplace R. The concentrations of calcium buffering proteins in mammalian cochlear hair cells. J Neurosci 2005;25:7867-75. DOI: https://doi.org/10.1523/JNEUROSCI.1196-05.2005
35. Krzywkowski P, Potier B, Billard JM, Dutar P, Lamour Y. Synaptic mechanisms and calcium binding proteins in the aged rat brain. Life Sci 1996;59:421-8. DOI: https://doi.org/10.1016/0024-3205(96)00321-9
36. Permyakov EA, Uversky VN. What is parvalbumin for? Biomolecules 2022;12:656. DOI: https://doi.org/10.3390/biom12050656
37. Yang D, Thalmann I, Thalmann R, Simmons DD. Expression of alpha and beta parvalbumin is differentially regulated in the rat organ of Corti during development. J Neurobiol 2004;58:479-92. DOI: https://doi.org/10.1002/neu.10289
38. Soto-Prior A, Cluzel M, Renard N, Ripoll C, Lavigne-Rebillard M, Eybalin M, Hamel CP. Molecular cloning and expression of alpha parvalbumin in the guinea pig cochlea. Brain Res Mol Brain Res 1995;34:337-42. DOI: https://doi.org/10.1016/0169-328X(95)00205-7
39. Simmons DD, Tong B, Schrader AD, Hornak AJ. Oncomodulin identifies different hair cell types in the mammalian inner ear. J Comp Neurol 2010;518:3785-802. DOI: https://doi.org/10.1002/cne.22424
40. Lv J, Wang H, Cheng X, Chen Y, Wang D, Zhang L, et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet 2024;403:2317-25. DOI: https://doi.org/10.1016/S0140-6736(23)02874-X
41. Qi J, Tan F, Zhang L, Lu L, Zhang S, Zhai Y, et al. AAV-mediated gene therapy restores hearing in patients with DFNB9 deafness. Adv Sci (Weinh) 2024;11:e2306788. DOI: https://doi.org/10.1002/advs.202306788
42. Akil O, Dyka F, Calvet C, Emptoz A, Lahlou G, Nouaille S, et al. Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model. Proc Natl Acad Sci USA 2019;116:4496-501. DOI: https://doi.org/10.1073/pnas.1817537116
43. Leclère JC, Dulon D. Otoferlin as a multirole Ca(2+) signaling protein: from inner ear synapses to cancer pathways. Front Cell Neurosci 2023;17:1197611. DOI: https://doi.org/10.3389/fncel.2023.1197611
44. Manchanda A, Chatterjee P, Bonventre JA, Haggard DE, Kindt KS, Tanguay RL, Johnson CP. Otoferlin depletion results in abnormal synaptic ribbons and altered intracellular calcium levels in zebrafish. Sci Rep 2019;9:14273. DOI: https://doi.org/10.1038/s41598-019-50710-2
45. Chatterjee P, Padmanarayana M, Abdullah N, Holman CL, LaDu J, Tanguay RL, Johnson CP. Otoferlin deficiency in zebrafish results in defects in balance and hearing: rescue of the balance and hearing phenotype with full-length and truncated forms of mouse otoferlin. Mol Cell Biol 2015;35:1043-54. DOI: https://doi.org/10.1128/MCB.01439-14
46. Ramakrishnan NA, Drescher MJ, Morley BJ, Kelley PM, Drescher DG. Calcium regulates molecular interactions of otoferlin with soluble NSF attachment protein receptor (SNARE) proteins required for hair cell exocytosis. J Biol Chem 2014;289:8750-66. DOI: https://doi.org/10.1074/jbc.M113.480533
47. Duncker SV, Franz C, Kuhn S, Schulte U, Campanelli D, Brandt N, et al. Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells. J Neurosci 2013;33:9508-19. DOI: https://doi.org/10.1523/JNEUROSCI.5689-12.2013
48. Schug N, Braig C, Zimmermann U, Engel J, Winter H, Ruth P, et al. Differential expression of otoferlin in brain, vestibular system, immature and mature cochlea of the rat. Eur J Neurosci 2006;24:3372-80. DOI: https://doi.org/10.1111/j.1460-9568.2006.05225.x
49. Liu W, Wang C, Yu H, Liu S, Yang J. Expression of acetylated tubulin in the postnatal developing mouse cochlea. Eur J Histochem 2018;62:2942. DOI: https://doi.org/10.4081/ejh.2018.2942
51. Liu WJ, Yang J. Developmental expression of inositol 1, 4, 5-trisphosphate receptor in the post-natal rat cochlea. Eur J Histochem 2015;59:2486. DOI: https://doi.org/10.4081/ejh.2015.2486
52. Minowa O, Ikeda K, Sugitani Y, Oshima T, Nakai S, Katori Y, et al. Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness. Science 1999;285:1408-11. DOI: https://doi.org/10.1126/science.285.5432.1408
53. Sadanandan J, Sathyanesan M, Newton SS. Aging alters the expression of trophic factors and tight junction proteins in the mouse choroid plexus. Fluids Barriers CNS 2024;21:77. DOI: https://doi.org/10.1186/s12987-024-00574-0
54. Rose KP, Manilla G, Milon B, Zalzman O, Song Y, Coate TM, Hertzano R. Spatially distinct otic mesenchyme cells show molecular and functional heterogeneity patterns before hearing onset. iScience 2023;26:107769. DOI: https://doi.org/10.1016/j.isci.2023.107769
55. Lopez IA, Zhao PM, Yamaguchi M, de Vellis J, Espinosa-Jeffrey A. Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse. Int J Dev Neurosci 2004;22:205-13. DOI: https://doi.org/10.1016/j.ijdevneu.2004.04.006
56. Malgrange B, Belachew S, Thiry M, Nguyen L, Rogister B, Alvarez ML, et al. Proliferative generation of mammalian auditory hair cells in culture. Mech Dev 2002;112:79-88. DOI: https://doi.org/10.1016/S0925-4773(01)00642-6
57. Martone T, Giordano P, Dagna F, Carulli D, Albera R, Rossi F. Nestin expression and reactive phenomena in the mouse cochlea after kanamycin ototoxicity. Eur J Neurosci 2014;39:1729-41. DOI: https://doi.org/10.1111/ejn.12576
58. Watanabe R, Morell MH, Miller JM, Kanicki AC, O'Shea KS, Altschuler RA, Raphael Y. Nestin-expressing cells in the developing, mature and noise-exposed cochlear epithelium. Mol Cell Neurosci 2012;49:104-9. DOI: https://doi.org/10.1016/j.mcn.2011.11.001
59. Mokrý J, Ehrmann J, Karbanová J, Cízková D, Soukup T, Suchánek J, et al. Expression of intermediate filament nestin in blood vessels of neural and non-neural tissues. Acta Medica (Hradec Kralove) 2008;51:173-9. DOI: https://doi.org/10.14712/18059694.2017.20
60. Mokrý J, Cízková D, Filip S, Ehrmann J, Osterreicher J, Kolár Z, English D. Nestin expression by newly formed human blood vessels. Stem Cells Dev 2004;13:658-64. DOI: https://doi.org/10.1089/scd.2004.13.658
61. Majesky MW. Vascular development. Arterioscler Thromb Vasc Biol 2018;38:e17-e24. DOI: https://doi.org/10.1161/ATVBAHA.118.310223
62. Suzuki S, Namiki J, Shibata S, Mastuzaki Y, Okano H. The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells, but not in mature vasculature. J Histochem Cytochem 2010;58:721-30. DOI: https://doi.org/10.1369/jhc.2010.955609
63. Klein D, Meissner N, Kleff V, Jastrow H, Yamaguchi M, Ergün S, Jendrossek V. Nestin(+) tissue-resident multipotent stem cells contribute to tumor progression by differentiating into pericytes and smooth muscle cells resulting in blood vessel remodeling. Front Oncol 2014;4:169. DOI: https://doi.org/10.3389/fonc.2014.00169
64. Onisim A, Achimas-Cadariu A, Vlad C, Kubelac P, Achimas-Cadariu P. Current insights into the association of Nestin with tumor angiogenesis. J BUON 2015;20:699-706.
65. Schwaller B. Cytosolic Ca2+ buffers. Cold Spring Harb Perspect Biol 2010;2:a004051. DOI: https://doi.org/10.1101/cshperspect.a004051
66. Yang Y, Murtha K, Climer LK, Ceriani F, Thompson P, Hornak AJ, et al. Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells. J Physiol 2023;601:4291-308. DOI: https://doi.org/10.1113/JP284690
67. Murtha KE, Yang Y, Ceriani F, Jeng JY, Climer LK, Jones F, et al. Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells. Cell Calcium 2022;105:102613. DOI: https://doi.org/10.1016/j.ceca.2022.102613
68. Spatz WB, Löhle E. Calcium-binding proteins in the spiral ganglion of the monkey, Callithrix jacchus. Hear Res 1995;86:89-99. DOI: https://doi.org/10.1016/0378-5955(95)00059-D
69. Fischer N, Johnson Chacko L, Majerus A, Potrusil T, Riechelmann H, Schmutzhard J, et al. Age-dependent calcium-binding protein expression in the spiral ganglion and hearing performance of C57BL/6J and 129/SvJ mice. ORL J Otorhinolaryngol Relat Spec 2019;81:138-54. DOI: https://doi.org/10.1159/000499472
70. Levic S, Bouleau Y, Dulon D. Developmental acquisition of a rapid calcium-regulated vesicle supply allows sustained high rates of exocytosis in auditory hair cells. PLoS One 2011;6:e25714. DOI: https://doi.org/10.1371/journal.pone.0025714
71. Takago H, Oshima-Takago T, Moser T. Disruption of otoferlin alters the mode of exocytosis at the mouse inner hair cell ribbon synapse. Front Mol Neurosci 2019;11:492. DOI: https://doi.org/10.3389/fnmol.2018.00492
72. Heidrych P, Zimmermann U, Kuhn S, Franz C, Engel J, Duncker SV, et al. Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell. Hum Mol Genet 2009;18:2779-90. DOI: https://doi.org/10.1093/hmg/ddp213
73. Dulon D, Safieddine S, Jones SM, Petit C. Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses. J Neurosci 2009;29:10474-87. DOI: https://doi.org/10.1523/JNEUROSCI.1009-09.2009
74. Conrad LJ, Grandi FC, Carlton AJ, Jeng JY, Tomasi L, Zarecki P, et al. The upregulation of K+ and HCN channels in developing spiral ganglion neurons is mediated by cochlear inner hair cells. The upregulation of K+ and HCN channels in developing spiral ganglion neurons is mediated by cochlear inner hair cells. J Physiol 2024;602:5329-51. DOI: https://doi.org/10.1113/JP286134
75. Beurg M, Safieddine S, Roux I, Bouleau Y, Petit C, Dulon D. Calcium- and otoferlin-dependent exocytosis by immature outer hair cells. J Neurosci 2008;28:1798-803. DOI: https://doi.org/10.1523/JNEUROSCI.4653-07.2008
76. Sundaresan S, Balasubbu S, Mustapha M. Thyroid hormone is required for the pruning of afferent type II spiral ganglion neurons in the mouse cochlea. Neuroscience 2016;312:165-78. DOI: https://doi.org/10.1016/j.neuroscience.2015.11.020

Ethics Approval

all animal studies, including the euthanasia procedure, were authorized by the Institutional Animal Care and Use Committee (IACUC) of Southeast University (approval No. 20200402025)

Supporting Agencies

this work was supported by the National Natural Science Foundation of China (No. 82000987) and the Natural Science Foundation of Jiangsu Province (No. BK20200394)

How to Cite



1.
Liu W, Zhang Y, Liang C, Yang S. Expression of nestin, parvalbumin and otoferlin during cochlear development in the mouse: an immunofluorescence study. Eur J Histochem [Internet]. 2025 Sep. 22 [cited 2025 Dec. 28];69(4). Available from: https://www.ejh.it/ejh/article/view/4242
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