Characterization of Adult Vestibular Organs in 11 CreER Mouse Lines

  • Jennifer S. Stone
  • Serena R. Wisner
  • Stephanie A. Bucks
  • Marcia M. Mellado Lagarde
  • Brandon C. CoxEmail author
Research Article


Utricles are vestibular sense organs that encode linear head movements. They are composed of a sensory epithelium with type I and type II hair cells and supporting cells, sitting atop connective tissue, through which vestibular nerves project. We characterized utricular Cre expression in 11 murine CreER lines using the ROSA26tdTomato reporter line and tamoxifen induction at 6 weeks of age. This characterization included Calbindin2CreERT2, Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, ParvalbuminCreERT2, Prox1CreERT2, Sox2CreERT2, and Sox9-CreERT2. OtoferlinCreERT2 mice had inducible Cre activity specific to hair cells. GLAST-CreERT2, Id2CreERT2, and Sox9-CreERT2 had inducible Cre activity specific to supporting cells. Sox2CreERT2 had inducible Cre activity in supporting cells and most type II hair cells. ParvalbuminCreERT2 mice had small numbers of labeled vestibular nerve afferents. Calbindin2CreERT2 mice had labeling of most type II hair cells and some type I hair cells and supporting cells. Only rare (or no) tdTomato-positive cells were detected in utricles of Fgfr3-iCreERT2, GFAP-A-CreER™, GFAP-B-CreER™, and Prox1CreERT2 mice. No Cre leakiness (tdTomato expression in the absence of tamoxifen) was observed in OtoferlinCreERT2 mice. A small degree of leakiness was seen in GLAST-CreERT2, Id2CreERT2, Sox2CreERT2, and Sox9-CreERT2 lines. Calbindin2CreERT2 mice had similar tdTomato expression with or without tamoxifen, indicating lack of inducible control under the conditions tested. In conclusion, 5 lines—GLAST-CreERT2, Id2CreERT2, OtoferlinCreERT2, Sox2CreERT2, and Sox9-CreERT2—showed cell-selective, inducible Cre activity with little leakiness, providing new genetic tools for researchers studying the vestibular periphery.


CreER/loxP mouse genetics utricle fate-mapping calbindin FGFR3 GFAP GLAST Id2 otoferlin parvalbumin Prox1 Sox2 Sox9 



This work was supported by the National Institutes of Health (F32 DC013695 to SAB, R01 DC013771 to JSS, R01 DC03696 to JSS, R01 DC014441 to BCC, and P30 DC04661 to the UW Research Core Center), the Office of Naval Research (N00014-13-1-0569 to BCC), the Office of the Assistant Secretary of Defense for Health Affairs (W81XWH-15-1-0475 to BCC), and the Wellcome Trust (089015 to MML). We thank Tot Nguyen, Irina Omelchenko, and Jialin Shang from the University of Washington, and Michelle Randle and Kaley Graves from Southern Illinois University School of Medicine, for technical assistance. We are grateful to Dr. Suzanne Baker (St. Jude Children’s Research Hospital) for sharing GFAP-A-CreER™ and GFAP-B-CreER™ mice; Dr. Ulrich Müller (The Scripps Research Institute) for sharing OtoferlinCreERT2 mice; Dr. Guillermo Oliver (St. Jude Children’s Research Hospital) for sharing Prox1CreERT2 mice; and Dr. William Richardson (University College London) for sharing Fgfr3-iCreERT2 mice. We thank Dr. Jian Zuo (St. Jude Children’s Research Hospital) for his support of the project which began when Marcia Mellado Lagarde and Brandon Cox were postdoctoral fellows in his lab.

Compliance with Ethical Standards

Conflict of Interest

Brandon C. Cox is a consultant with Turner Scientific, LLC. No other authors have any professional or financial affiliations that may be perceived as a conflict of interest.


  1. Arnold K, Sarkar A, Yram MA, Polo JM, Bronson R (2011) Sox2+ adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 9(4):317–329PubMedPubMedCentralCrossRefGoogle Scholar
  2. Atkinson PJ, Dong Y, Gu S, Liu W, Udagawa T, Cheng AG (2018) Sox2 haploinsufficiency primes regeneration and Wnt-responsiveness in the mouse cochlea. J Clin Invest in pressGoogle Scholar
  3. Bermingham-McDonogh O, Stone JS, Reh TA, Rubel EW (2001) FGFR3 expression during development and regeneration of the chick inner ear sensory epithelia. Dev Biol 238(2):247–259PubMedCrossRefGoogle Scholar
  4. Bermingham-McDonogh O, Oesterle EC, Stone JS, Hume CR, Huynh HM, Hayashi T (2006) Expression of Prox1 during mouse cochlear development. J Comp Neurol 496(2):172–186PubMedPubMedCentralCrossRefGoogle Scholar
  5. Bucks SA, Cox BC, Vlosich BA, Manning JP, Nguyen TB, Stone JS (2017) Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice. elife 6:e18128PubMedPubMedCentralCrossRefGoogle Scholar
  6. Buelow B, Scharenberg AM (2008) Characterization of parameters required for effective use of tamoxifen-regulated recombination. Edited by Sebastian D. Fugmann. PLoS One 3(9):e3264PubMedPubMedCentralCrossRefGoogle Scholar
  7. Burns JC, Cox BC, Thiede BR, Zuo J, Corwin JT (2012a) In vivo proliferative regeneration of balance hair cells in newborn mice. J Neurosci 32(19):6570–6577PubMedPubMedCentralCrossRefGoogle Scholar
  8. Burns JC, On D, Baker W, Collado MS, Corwin JT (2012b) Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and Striolar growth zones. J Ass Res Otolaryngol 13(5):609–627CrossRefGoogle Scholar
  9. Cafaro J, Lee GS, Stone JS (2007) Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration. Dev Dyn 236(1):156–170PubMedCrossRefGoogle Scholar
  10. Chow LM, Zhang J, Baker SJ (2008) Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells. Transgenic Res 17(5):919–928PubMedPubMedCentralCrossRefGoogle Scholar
  11. Cochrane RL, Clark SH, Harris A, Kream BE (2007) Rearrangement of a conditional allele regardless of inheritance of a Cre recombinase transgene. Genesis 45(1):17–20PubMedCrossRefGoogle Scholar
  12. Cox BC, Liu Z, Mellado Lagarde MM, Zuo J (2012) Conditional gene expression in the mouse inner ear using Cre-loxP. J Ass Res Otolaryngol 13(3):295–322CrossRefGoogle Scholar
  13. Cox BC, Chai R, Lenoir A, Liu Z, Zhang L, Nguyen D-H, Chalasani K, Steigelman KA, Fang J, Rubel EW, Cheng AG, Zuo J (2014) Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo. Development 141(4):816–829PubMedPubMedCentralCrossRefGoogle Scholar
  14. Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8(24):1323–13S2CrossRefGoogle Scholar
  15. Dechesne CJ, Winsky L, Kim HN, Goping G, Vu TD, Wenthold RJ, Jacobowitz DM (1991) Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear. Brain Res 560(1–2):139–148PubMedCrossRefGoogle Scholar
  16. Demêmes D, Eybalin M, Renard N (1993) Cellular distribution of parvalbumin immunoreactivity in the peripheral vestibular system of three rodents. Cell Tissue Res 274(3):487–492PubMedCrossRefGoogle Scholar
  17. Desai SS, Zeh C, Lysakowski A (2005) Comparative morphology of rodent vestibular periphery. I. Saccular and utricular maculae. J Neurophysiol 93(1):251–266PubMedCrossRefGoogle Scholar
  18. Dulon D, Safieddine S, Jones SM, Petit C (2009) Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses. J Neurosci 29(34):10474–10487PubMedPubMedCentralCrossRefGoogle Scholar
  19. Dvorakova M, Jahan I, Macova I, Chumak T, Bohuslavova R, Syka J, Fritzsch B, Pavlinkova G (2016) Incomplete and delayed Sox2 deletion defines residual ear neurosensory development and maintenance. Sci Rep 6(1):38253PubMedPubMedCentralCrossRefGoogle Scholar
  20. Eatock RA, Songer JE (2011) Vestibular hair cells and afferents: two channels for head motion signals. Annu Rev Neurosci 34:501–534PubMedCrossRefGoogle Scholar
  21. Elsir T, Smits A, Lindström MS, Nistér M (2012) Transcription factor PROX1: its role in development and cancer. Cancer Metastasis Rev 31(3–4):793–805PubMedCrossRefGoogle Scholar
  22. Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P (1996) Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A 93(20):10887–10890PubMedPubMedCentralCrossRefGoogle Scholar
  23. Fritzsch B, Pauley S, Beisel KW (2006) Cells, molecules and morphogenesis: the making of the vertebrate ear. Brain Res 1091(1):151–171PubMedPubMedCentralCrossRefGoogle Scholar
  24. Gallardo T, Shirley L, John GB, Castrillon DH (2007) Generation of a germ cell-specific mouse transgenic Cre line, vasa-Cre. Genesis 45(6):413–417PubMedPubMedCentralCrossRefGoogle Scholar
  25. Golub JS, Tong L, Ngyuen TB, Hume CR, Palmiter RD, Rubel EW, Stone JS (2012) Hair cell replacement in adult mouse utricles after targeted ablation of hair cells with diphtheria toxin. J Neurosci 32(43):15093–15105PubMedPubMedCentralCrossRefGoogle Scholar
  26. Gómez-Casati ME, Murtie J, Taylor B, Corfas G (2010) Cell-specific inducible gene recombination in postnatal inner ear supporting cells and glia. J Ass Res Otolaryngol 11(1):19–26CrossRefGoogle Scholar
  27. Hartman BH, Basak O, Nelson BR, Taylor V, Bermingham-Mcdonogh O, Reh TA (2009) Hes5 expression in the postnatal and adult mouse inner ear and the drug-damaged cochlea. J Ass Res Otolaryngol 10(3):321–340CrossRefGoogle Scholar
  28. Hartman BH, Reh TA, Bermingham-McDonogh O (2010) Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear. Proc Natl Acad Sci U S A 107(36):15792–15797PubMedPubMedCentralCrossRefGoogle Scholar
  29. Hasson T, Mooseker MS (1997) The growing family of myosin motors and their role in neurons and sensory cells. Curr Opin Neurobiol 7(5):615–623PubMedCrossRefGoogle Scholar
  30. Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244(2):305–318PubMedPubMedCentralCrossRefGoogle Scholar
  31. Hayashi S, Tensen T, McMahon AP (2003) Maternal inheritance of Cre activity in a Sox2Cre deleter strain. Genesis 37(2):51–53PubMedCrossRefGoogle Scholar
  32. Hayashi T, Cunningham D, Bermingham-McDonogh O (2007) Loss of FGFR3 leads to excess hair cell development in the mouse organ of corti. Devl Dyn 236(2):525–533CrossRefGoogle Scholar
  33. Heffner CS, Herbert Pratt C, Babiuk RP, Sharma Y, Rockwood SF, Donahue LR, Eppig JT, Murray SA (2012) Supporting conditional mouse mutagenesis with a comprehensive Cre characterization resource. Nat Commun 3:1218PubMedPubMedCentralCrossRefGoogle Scholar
  34. Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the Tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 27(22):4324–4327PubMedPubMedCentralCrossRefGoogle Scholar
  35. Jaenisch R, Jähner D, Nobis P, Simon I, Löhler J, Harbers K, Grotkopp D (1981) Chromosomal position and activation of retroviral genomes inserted into the germ line of mice. Cell 24(2):519–529PubMedCrossRefGoogle Scholar
  36. Jones JM, Montcouquiol M, Dabdoub A, Woods C, Kelley MW (2006) Inhibitors of differentiation and DNA binding (ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 26(2):550–558PubMedPubMedCentralCrossRefGoogle Scholar
  37. 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(7036):1031–1035PubMedCrossRefGoogle Scholar
  38. Kirjavainen A, Sulg M, Heyd F, Alitalo K, Ylä-Herttuala S, Möröy T, Petrova TV, Pirvola U (2008) Prox1 interacts with Atoh1 and Gfi1, and regulates cellular differentiation in the inner ear sensory epithelia. Devl Biol 322(1):33–45CrossRefGoogle Scholar
  39. Kopp JL, Dubois CL, Schaffer AE, Hao E, Shih HP, Seymour PA, Ma J, Sander M (2011) Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Development 138(4):653–665PubMedPubMedCentralCrossRefGoogle Scholar
  40. Ku Y-C, Renaud NA, Veile RA, Helms C, Voelker CCJ, Warchol ME, Lovett M (2014) The transcriptome of utricle hair cell regeneration in the avian inner ear. J Neurosci 34(10):3523–3535PubMedPubMedCentralCrossRefGoogle Scholar
  41. Kwan KM (2002) Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32(2):49–62PubMedCrossRefGoogle Scholar
  42. Lin J, Zhang X, Wu F, Lin W (2015) Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle. Front Cell Neurosci 9:113PubMedPubMedCentralGoogle Scholar
  43. Ling F, Kang B, Sun X-H (2014) Id proteins: small molecules, mighty regulators. Curr Top Devl Biol 110:189–216CrossRefGoogle Scholar
  44. Lomeli H, Ramos-Mejia V, Gertsenstein M, Lobe CG, Nagy A (2000) Targeted insertion of Cre recombinase into the TRAP gene:excision in primordial germ cells. Genesis 26(2):116–117PubMedCrossRefGoogle Scholar
  45. Loponen H, Ylikoski J, Albrecht JH, Pirvola U (2011) Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression. PLoS One 6(11):e27360PubMedPubMedCentralCrossRefGoogle Scholar
  46. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13(1):133–140PubMedCrossRefGoogle Scholar
  47. Moser T, Starr A (2016) Auditory neuropathy—neural and synaptic mechanisms. Nat Rev Neurol 12(3):135–149PubMedCrossRefGoogle Scholar
  48. Nakamura E, Nguyen M-T, Mackem S (2006) Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Dev Dyn 235(9):2603–2612PubMedCrossRefGoogle Scholar
  49. Nouvian R, Beutner D, Parsons TD, Moser T (2006) Structure and function of the hair cell ribbon synapse. J Memb Biol 209(2–3):153–165CrossRefGoogle Scholar
  50. Oesterle EC, Campbell S, Taylor RR, Forge A, Hume CR (2008) Sox2 and Jagged1 expression in normal and drug-damaged adult mouse inner ear. J Ass Res Otolaryngol 9(1):65–89CrossRefGoogle Scholar
  51. Pekny M, Pekna M (2004) Astrocyte intermediate filaments in CNS pathologies and regeneration. J Pathol 204(4):428–437PubMedCrossRefGoogle Scholar
  52. Pujol R, Pickett SB, Nguyen TB, Stone JS (2014) Large basolateral processes on type II hair cells are novel processing units in mammalian vestibular organs. J Comp Neurol 522(14):3141–3159PubMedPubMedCentralCrossRefGoogle Scholar
  53. Rawlins EL, Clark CP, Xue Y, Hogan BLM (2009) The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development 136(22):3741–3745PubMedPubMedCentralCrossRefGoogle Scholar
  54. Raymond J, Dechesne CJ, Desmadryl G, Dememes D (1993) Different calcium-binding proteins identify subpopulations of vestibular ganglion neurons in the rat. Acta Oto-Laryngol Supp 503:114–118CrossRefGoogle Scholar
  55. Rio C, Dikkes P, Liberman MC, Corfas G (2002) Glial fibrillary acidic protein expression and promoter activity in the inner ear of developing and adult mice. J Comp Neurol 442(2):156–162PubMedCrossRefGoogle Scholar
  56. Rivers LE, Young KM, Rizzi M, Jamen F, Psachoulia K, Wade A, Kessaris N, Richardson WD (2008) PDGFRA/NG2 glia generate myelinating oligodendrocytes and piriform projection neurons in adult mice. Nat Neurosci 11(12):1392–1401PubMedCrossRefGoogle Scholar
  57. Robinson SP, Langan-Fahey SM, Johnson DA, Jordan VC (1991) Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient. Drug Metab Dispos 19(1):36–43PubMedGoogle Scholar
  58. Romero-Carvajal A, Acedo JN, Jiang L, Kozlovskaja-Gumbriene A, Alexander R, Li H, Piotrowski T (2015) Regeneration of sensory hair cells requires localized interactions between the notch and Wnt pathways. Devl Cell 34(3):267–282CrossRefGoogle Scholar
  59. Roux I, Safieddine S, Nouvian R, Grati M, Simmler M-C, Bahloul A, Perfettini I et al (2006) Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell 127(2):277–289PubMedCrossRefGoogle Scholar
  60. Rüsch A, Eatock RA (1996) A delayed rectifier conductance in type I hair cells of the mouse utricle. J Neurophysiol 76(2):995–1004PubMedCrossRefGoogle Scholar
  61. Rüsch A, Lysakowski A, Eatock RA (1998) Postnatal development of type I and type II hair cells in the mouse utricle: acquisition of voltage-gated conductances and differentiated morphology. J Neurosci 18(18):7487–7501PubMedCrossRefGoogle Scholar
  62. Schug N, Braig C, Zimmermann U, Engel J, Winter H, Ruth P, Blin N, Pfister M, Kalbacher H, Knipper M (2006) Differential expression of otoferlin in brain, vestibular system, immature and mature cochlea of the rat. Eur J Neurosci 24(12):3372–3380PubMedCrossRefGoogle Scholar
  63. Siegenthaler JA, Tremper-Wells BA, Miller MW (2008) Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression. Cereb Cortex 18(8):1865–1875PubMedCrossRefGoogle Scholar
  64. Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, Samokhvalov IM, Oliver G (2007) Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev 21(19):2422–2432PubMedPubMedCentralCrossRefGoogle Scholar
  65. Takayasu Y, Iino M, Takatsuru Y, Tanaka K, Ozawa S (2009) Functions of glutamate transporters in cerebellar Purkinje cell synapses. Acta Physiol 197(1):1–12CrossRefGoogle Scholar
  66. Takumi Y, Matsubara A, Danbolt NC, Laake JH, Storm-Mathisen J, Usami S, Shinkawa H, Ottersen OP (1997) Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ. Neuroscience 79(4):1137–1144PubMedCrossRefGoogle Scholar
  67. Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K, Kvitsani D, Fu Y, Lu J, Lin Y, Miyoshi G, Shima Y, Fishell G, Nelson SB, Huang ZJ (2011) A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71(6):995–1013PubMedPubMedCentralCrossRefGoogle Scholar
  68. Wang Y, Rattner A, Zhou Y, Williams J, Smallwood PM, Nathans J (2012) Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity. Cell 151(6):1332–1344PubMedPubMedCentralCrossRefGoogle Scholar
  69. Wang T, Chai R, Kim GS, Pham N, Jansson L, Nguyen D-H, Kuo B, May LA, Zuo J, Cunningham LL, Cheng AG (2015) Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle. Nat Commun 6:6613PubMedPubMedCentralCrossRefGoogle Scholar
  70. Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6(1):679–714PubMedCrossRefGoogle Scholar
  71. Wu J, Li W, Lin C, Chen Y, Cheng C, Sun S, Tang M, Chai R, Li H (2016) Co-regulation of the notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles. Sci Rep 6(1):29418PubMedPubMedCentralCrossRefGoogle Scholar
  72. Young KM, Mitsumori T, Pringle N, Grist M, Kessaris N, Richardson WD (2010) An Fgfr3-iCreERT2 transgenic mouse line for studies of neural stem cells and astrocytes. Glia 58(8):943–953PubMedPubMedCentralGoogle Scholar

Copyright information

© Association for Research in Otolaryngology 2018

Authors and Affiliations

  • Jennifer S. Stone
    • 1
  • Serena R. Wisner
    • 1
  • Stephanie A. Bucks
    • 1
  • Marcia M. Mellado Lagarde
    • 2
  • Brandon C. Cox
    • 3
    Email author
  1. 1.Department of Otolaryngology-Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research CenterUniversity of WashingtonSeattleUSA
  2. 2.Department of Developmental NeurobiologySt. Jude Children’s Research HospitalMemphisUSA
  3. 3.Departments of Pharmacology and Surgery, Division of OtolaryngologySouthern Illinois University School of MedicineSpringfieldUSA

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