Abstract
Tsukushi is a small, leucine-rich repeat proteoglycan that interacts with and regulates essential cellular signaling cascades in the chick retina and murine subventricular zone, hippocampus, dermal hair follicles, and the cochlea. However, its function in the vestibules of the inner ear remains unknown. Here, we investigated the function of Tsukushi in the vestibules and found that Tsukushi deficiency in mice resulted in defects in posterior semicircular canal formation in the vestibules, but did not lead to vestibular hair cell loss. Furthermore, Tsukushi accumulated in the non-prosensory and prosensory regions during the embryonic and postnatal developmental stages. The downregulation of Tsukushi altered the expression of key genes driving vestibule differentiation in the non-prosensory regions. Our results indicate that Tsukushi interacts with Wnt2b, bone morphogenetic protein 4, fibroblast growth factor 10, and netrin 1, thereby controlling semicircular canal formation. Therefore, Tsukushi may be an essential component of the molecular pathways regulating vestibular development.
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References
Ahmad SAI, Anam MB, Ito N, Ohta K (2018) Involvement of Tsukushi in diverse developmental processes. J Cell Commun Signal 12:205–210. https://doi.org/10.1007/s12079-018-0452-8
Ahmad SAI, Anam MB, Istiaq A, Ito N, Ohta K (2020) Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells. Dev Growth Differ 62:108–117. https://doi.org/10.1111/dgd.12649
Alsina B, Whitfield TT (2017) Sculpting the labyrinth: morphogenesis of the developing inner ear. Semin Cell Dev Biol 65:47–59. https://doi.org/10.1016/j.semcdb.2016.09.015
Baynash AG, Hosoda K, Giaid A, Richardson JA, Emoto N, Hammer RE, Yanagisawa M (1994) Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79:1277–1285. https://doi.org/10.1016/0092-8674(94)90018-3
Bovolenta P, Esteve P, Ruiz JM, Cisneros E, Lopez-Rios J (2008) Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J Cell Sci 121:737–746. https://doi.org/10.1242/jcs.026096
Chang W, Lin Z, Kulessa H, Hebert J, Hogan BLM, Wu DK (2008) Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements. PLOS Genet 4:e1000050. https://doi.org/10.1371/journal.pgen.1000050
Dabdoub A, Puligilla C, Jones JM, Fritzsch B, Cheah KSE, Pevny LH, Kelley MW (2008) SOX2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proc Natl Acad Sci USA 105:18396–18401. https://doi.org/10.1073/pnas.0808175105
Dellett M, Hu W, Papadaki V, Ohnuma S (2012) Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance. Dev Growth Differ 54:327–340. https://doi.org/10.1111/j.1440-169X.2012.01339.x
Di Lella F, Falcioni M, Piazza P (2007) Dehiscence of posterior semicircular canal. Otol Neurotol 28:280–281. https://doi.org/10.1097/01.mao.0000231592.64972.5b
Fekete DM, Homburger SA, Waring MT, Riedl AE, Garcia LF (1997) Involvement of programmed cell death in morphogenesis of the vertebrate inner ear. Development 124:2451–2461. https://doi.org/10.1242/dev.124.12.2451
Fernagut PO, Diguet E, Labattu B, Tison F (2002) A simple method to measure stride length as an index of nigrostriatal dysfunction in mice. J Neurosci Methods 113:123–130. https://doi.org/10.1016/S0165-0270(01)00485-X
Higashi K, Matsuki C, Sarashina N (1992) Aplasia of posterior semicircular canal in Waardenburg syndrome type II. J Otolaryngol 21:262–264
Hocking AM, Shinomura T, McQuillan DJ (1998) Leucine-rich repeat glycoproteins of the extracellular matrix. Matrix Biol 17:1–19. https://doi.org/10.1016/S0945-053X(98)90121-4
Hossain M, Ahmed G, Naser IB, Shinmyo Y, Ito A, Riyadh MA, Felemban A, Song X, Ohta K, Tanaka H (2013) The combinatorial guidance activities of draxin and Tsukushi are essential for forebrain commissure formation. Dev Biol 374:58–70. https://doi.org/10.1016/j.ydbio.2012.11.029
Huang SM, Mishina YM, Liu S et al (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461:614–620. https://doi.org/10.1038/nature08356
Irie K, Ogata H, Mitsudome A (1990) CT findings of the temporal bones in Waardenburg’s syndrome. No to Hattatsu 22:241–246
Ito A, Shinmyo Y, Abe T, Oshima N, Tanaka H, Ohta K (2010) Tsukushi is required for anterior commissure formation in mouse brain. Biochem Biophys Res Commun 402:813–818. https://doi.org/10.1016/j.bbrc.2010.10.127
Ito N, Riyadh MA, Ahmad SAI, Hattori S, Kanemura Y, Kiyonari H, Abe T, Furuta Y, Shinmyo Y, Kaneko N, Hirota Y, Lupo G, Hatakeyama J, Abdulhaleem MFA, Anam MB, Yamaguchi T, Takeo T, Takebayashi H, Takebayashi M, Oike Y, Nakagata N, Shimamura K, Holtzman MJ, Takahashi Y, Guillemot F, Miyakawa K, Sawamoto K, Ohta K (2021) Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone. Sci Transl Med 13:eaay7896
Jahan I, Pan N, Kersigo J, Fritzsch B (2013) Beyond generalized hair cells: molecular cues for hair cell types. Hear Res 297:30–41. https://doi.org/10.1016/j.heares.2012.11.008
Kempfle JS, Turban JL, Edge ASB (2016) SOX2 in the differentiation of cochlear progenitor cells. Sci Rep 6:23293. https://doi.org/10.1038/srep23293
Kiernan AE, Pelling AL, Leung KKH, Tang ASP, Bell DM, Tease C, Lovell-Badge R, Steel KP, Cheah KSE (2005) SOX2 is required for sensory organ development in the mammalian inner ear. Nature 434:1031–1035. https://doi.org/10.1038/nature03487
Kiernan AE, Xu J, Gridley T (2006) The notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLOS Genet 2:e4. https://doi.org/10.1371/journal.pgen.0020004
Lang H, Bever MM, Fekete DM (2000) Cell proliferation and cell death in the developing chick inner ear: spatial and temporal patterns. J Comp Neurol 417:205–220. https://doi.org/10.1002/(SICI)1096-9861(20000207)417:2%3c205::AID-CNE6%3e3.0.CO;2-Y
Langman J, Sadler TW (2008) Langman’s medical embryology, 12th edn. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia
Li H, Corrales CE, Wang Z, Zhao Y, Wang Y, Liu H, Heller S (2005) BMP4 signaling is involved in the generation of inner ear sensory epithelia. BMC Dev Biol 5:16. https://doi.org/10.1186/1471-213X-5-16
Martin P, Swanson GJ (1993) Descriptive and experimental analysis of the epithelial remodellings that control semicircular canal formation in the developing mouse inner ear. Dev Biol 159:549–558. https://doi.org/10.1006/dbio.1993.1263
Merline R, Schaefer RM, Schaefer L (2009) The matricellular functions of small leucine-rich proteoglycans (SLRPs). J Cell Commun Signal 3:323–335. https://doi.org/10.1007/s12079-009-0066-2
Miwa T, Minoda R, Ishikawa Y, Kajii T, Orita Y, Ohyama T (2019) Role of Dach1 revealed using a novel inner ear-specific Dach1-knockdown mouse model. Biol Open 8:bio043612. https://doi.org/10.1242/bio.043612
Miwa T, Ohta K, Ito N, Hattori S, Miyakawa T, Takeo T, Nakagata N, Song WJ, Minoda R (2020) Tsukushi is essential for the development of the inner ear. Mol Brain 13:29. https://doi.org/10.1186/s13041-020-00570-z
Morris SA, Almeida AD, Tanaka H, Ohta K, Ohnuma S (2007) Tsukushi modulates Xnr2:FGF and BMP signaling: regulation of Xenopus germ layer formation. PLoS ONE 2:e1004. https://doi.org/10.1371/journal.pone.0001004
Motohashi H, Hozawa K, Oshima T, Takeuchi T, Takasaka T (1994) Dysgenesis of melanocytes and cochlear dysfunction in mutant microphthalmia (mi) mice. Hear Res 80:10–20. https://doi.org/10.1016/0378-5955(94)90003-5
Ni GX, Li Z, Zhou YZ (2014) The role of small leucine-rich proteoglycans in osteoarthritis pathogenesis. Osteoarthr Cartil 22:896–903. https://doi.org/10.1016/j.joca.2014.04.026
Niimori D, Kawano R, Felemban A, Niimori-Kita K, Tanaka H, Ihn H, Ohta K (2012) Tsukushi controls the hair cycle by regulating TGF-β1 signaling. Dev Biol 372:81–87. https://doi.org/10.1016/j.ydbio.2012.08.030
Ohta K, Lupo G, Kuriyama S, Keynes R, Holt CE, Harris WA, Tanaka H, Ohnuma SI (2004) Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin. Dev Cell 7:347–358. https://doi.org/10.1016/j.devcel.2004.08.014
Ohta K, Kuriyama S, Okafuji T, Gejima R, Ohnuma S, Tanaka H (2006) Tsukushi cooperates with VG1 to induce primitive streak and Hensen’s node formation in the chick embryo. Development 133:3777–3786. https://doi.org/10.1242/dev.02579
Ohta K, Ito A, Kuriyama S, Lupo G, Kosaka M, Ohnuma SI, Nakagawa S, Tanaka H (2011) Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4. Proc Natl Acad Sci USA 108:14962–14967. https://doi.org/10.1073/pnas.1100513108
Ohuchi H, Yasue A, Ono K, Sasaoka S, Tomonari S, Takagi A, Itakura M, Moriyama K, Noji S, Nohno T (2005) Identification of cis-element regulating expression of the mouse Fgf10 gene during inner ear development. Dev Dyn 233:177–187. https://doi.org/10.1002/dvdy.20319
Ohyama T, Basch ML, Mishina Y, Lyons KM, Segil N, Groves AK (2010) BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea. J Neurosci 30:15044–15051. https://doi.org/10.1523/JNEUROSCI.3547-10.2010
Owens SE, Broman KW, Wiltshire T, Elmore JB, Bradley KM, Smith JR, Southard-Smith EM (2005) Genome-wide linkage identifies novel modifier loci of aganglionosis in the Sox10Dom model of Hirschsprung disease. Hum Mol Genet 14:1549–1558. https://doi.org/10.1093/hmg/ddi163
Pagedar NA, Wang W, Chen DHC, Davis RR, Lopez I, Wright CG, Alagramam KN (2006) Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection—a technical report based on preliminary findings. Brain Res 1091:289–299. https://doi.org/10.1016/j.brainres.2006.01.057
Pan W, Jin Y, Chen J, Rottier RJ, Steel KP, Kiernan AE (2013) Ectopic expression of activated notch or SOX2 reveals similar and unique roles in the development of the sensory cell progenitors in the mammalian inner ear. J Neurosci 33:16146–16157. https://doi.org/10.1523/JNEUROSCI.3150-12.2013
Pauley S, Wright TJ, Pirvola U, Ornitz D, Beisel K, Fritzsch B (2003) Expression and function of FGF10 in mammalian inner ear development. Dev Dyn 227:203–215. https://doi.org/10.1002/dvdy.10297
Pietraszek-Gremplewicz K, Karamanou K, Niang A, Dauchez M, Belloy N, Maquart FX, Baud S, Brézillon S (2019) Small leucine-rich proteoglycans and matrix metalloproteinase-14: key partners? Matrix Biol 75–76:271–285. https://doi.org/10.1016/j.matbio.2017.12.006
Postigo AA (2003) Opposing functions of ZEB proteins in the regulation of the TGFbeta/BMP signaling pathway. EMBO J 22:2443–2452. https://doi.org/10.1093/emboj/cdg225
Puligilla C, Dabdoub A, Brenowitz SD, Kelley MW (2010) SOX2 induces neuronal formation in the developing mammalian cochlea. J Neurosci 30:714–722. https://doi.org/10.1523/JNEUROSCI.3852-09.2010
Rakowiecki S, Epstein DJ (2013) Divergent roles for Wnt/β-catenin signaling in epithelial maintenance and breakdown during semicircular canal formation. Development 140:1730–1739. https://doi.org/10.1242/dev.092882
Rao J, Pfeiffer MJ, Frank S, Adachi K, Piccini I, Quaranta R, Araúzo-Bravo M, Schwarz J, Schade D, Leidel S, Schöler HR, Seebohm G, Greber B (2016) Stepwise clearance of repressive roadblocks drives cardiac induction in human ESCs. Cell Stem Cell 18:341–353. https://doi.org/10.1016/j.stem.2015.11.019
Salminen M, Meyer BI, Bober E, Gruss P (2000) Netrin 1 is required for semicircular canal formation in the mouse inner ear. Development 127:13–22
Schaefer L, Iozzo RV (2008) Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J Biol Chem 283:21305–21309. https://doi.org/10.1074/jbc.R800020200
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. https://doi.org/10.1038/nmeth.2089
Shibata S, Miwa T, Wu HH, Levitt P, Ohyama T (2016a) Hepatocyte growth factor–c-MET signaling mediates the development of nonsensory structures of the Mammalian Cochlea and hearing. J Neurosci 36:8200–8209. https://doi.org/10.1523/JNEUROSCI.4410-15.2016
Shibata SB, Ranum PT, Moteki H, Pan B, Goodwin AT, Goodman SS, Abbas PJ, Holt JR, Smith RJH (2016b) RNA interference prevents autosomal-dominant hearing loss. Am J Hum Genet 98:1101–1113. https://doi.org/10.1016/j.ajhg.2016.03.028
Takeda H, Miwa T, Kim MY, Choi BY, Orita Y, Minoda R (2019) Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function. Sci Rep 9:17979. https://doi.org/10.1038/s41598-019-54262-3
Uejima A, Amano T, Nomura N, Noro M, Yasue T, Shiroishi T, Ohta K, Yokoyama H, Tamura K (2010) Anterior shift in gene expression precedes anteriormost digit formation in amniote limbs. Dev Growth Differ 52:223–234. https://doi.org/10.1111/j.1440-169X.2009.01161.x
Vervoort R, Ceulemans H, Van Aerschot L, D’Hooge R, David G (2010) Genetic modification of the inner ear lateral semicircular canal phenotype of the Bmp4 haplo-insufficient mouse. Biochem Biophys Res Commun 394:780–785. https://doi.org/10.1016/j.bbrc.2010.03.069
Werb Z, Chin JR (1998) Extracellular matrix remodeling during morphogenesis. Ann NY Acad Sci 857:110–118. https://doi.org/10.1111/j.1749-6632.1998.tb10111.x
Wiener-Vacher SR, Amanou L, Denise P, Narcy P, Manach Y (1999) Vestibular function in children with the CHARGE association. Arch Otolaryngol Head Neck Surg 125:342–347. https://doi.org/10.1001/archotol.125.3.342
Wright CG, Brown OE, Meyerhoff WL, Rutledge JC (1985) Inner ear anomalies in two cases of trisomy 18. Am J Otolaryngol Neck Surg 6:392–404. https://doi.org/10.1016/S0196-0709(85)80018-1
Wu DK, Kelley MW (2012) Molecular mechanisms of inner ear development. Cold Spring Harb Perspect Biol 4:a008409. https://doi.org/10.1101/cshperspect.a008409
Yano K, Washio K, Tsumanuma Y, Yamato M, Ohta K, Okano T, Izumi Y (2017) The role of Tsukushi (TSK), a small leucine-rich repeat proteoglycan, in bone growth. Regen Ther 7:98–107. https://doi.org/10.1016/j.reth.2017.08.001
Zheng JL, Gao WQ (2000) Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 3:580–586. https://doi.org/10.1038/75753
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We thank Ms. Miho Kataoka for technical assistance and all laboratory members for their valuable help.
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TM performed the experiments, analyzed the data, and wrote the manuscript. TM, IN, and KO. designed the experiments and supervised the study.
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Miwa, T., Ito, N. & Ohta, K. Tsukushi is essential for the formation of the posterior semicircular canal that detects gait performance. J. Cell Commun. Signal. 15, 581–594 (2021). https://doi.org/10.1007/s12079-021-00627-1
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DOI: https://doi.org/10.1007/s12079-021-00627-1