BACKGROUND: Knockin mouse models expressing calbindin (Calb1), calretinin (Calb2), and peripherin (Prph) exhibit changes in hair cells (HCs), spiral ganglion neurons (SGN), vestibular ganglion neurons (VGNs), and their central projections. RESULTS: Developing cristae HCs show strong Calb1-positive expression, but adult HCs are mainly Calb2-positive. Utricle and saccule initially have Calb2-positive HCs and later develop Calb1-positive HCs in the striola region. Inner hair cells (IHCs) and outer hair cells (OHCs) in the cochlea express Calb2 early on. Calb1 expression in OHCs overlaps with Calb2; the expression of Myo7a, Calb1, and Calb2 reaches the apex later. SGNs and VGNs exhibit distinct Calb1 and Calb2 patterns but include a subpopulation with mixed expression. Central fibers are Calb1- and Calb2-positive early in the developing cochlear nuclei (CN) and vestibular nuclei (VN) but remain highly Prph-positive. VGNs innervate the lateral and VN, which are positive for Calb2 and Prph. Distinct Calb1-positive neurons overlap with the anterior (A) and ventral (V) cochlear nuclei (AVCN, PVCN) with Calb2, while the dorsal cochlear nucleus (DCN) shows segregation of Calb2 and Calb1. CONCLUSION: We offer insights into the timing of how neuronal identity and connectivity are regulated in the auditory and vestibular systems, as shown by the expression of Calb1, Calb2, and Prph.

Department of Biology The University of Iowa Iowa City Iowa USA

Department of Neurological Sciences University of Nebraska Medical Center Omaha Nebraska USA

Department of Translational Neurosciences College of Medicine University of Arizona Phoenix Arizona USA

Laboratory of Molecular Pathogenetics Institute of Biotechnology CAS BIOCEV Center of Excellence Vestec Czechia

Zobrazit více v PubMed

Oertel D, Cao X‐J. 2.27—the ventral Cochlear nucleus. In: Fritzsch B, ed. The Senses: A Comprehensive Reference (Second Edition). Elsevier; 2020:517‐532.

Grothe B. 2.01—the Auditory system function ‐ an integrative perspective. In: Fritzsch B, ed. The Senses: A Comprehensive Reference (Second Edition). Elsevier; 2020:1‐17.

Zine A, Fritzsch B. Early steps towards hearing: placodes and sensory development. Int J Mol Sci. 2023;24(8):6994. https://doi.org10.3390/ijms24086994

Pyott SJ, Pavlinkova G, Yamoah EN, Fritzsch B. Harmony in the molecular orchestra of hearing: developmental mechanisms from the ear to the brain. Annu Rev Neurosci. 2024;47(1):1‐20. https://doi.org10.1146/annurev-neuro-081423-093942

Schwaller B. Cytosolic Ca2+ buffers are inherently Ca2+ signal modulators. Cold Spring Harb Perspect Biol. 2020;12(1):a035543.

Climer LK, Cox AM, Reynolds TJ, Simmons DD. Oncomodulin: the enigmatic parvalbumin protein. Front Mol Neurosci. 2019;12:235.

Yang Y, Murtha K, Climer LK, et al. Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells. J Physiol. 2023;601(19):4291‐4308.

Kevetter GA, Leonard RB. Molecular probes of the vestibular nerve. II. Characterization of neurons in Scarpa's ganglion to determine separate populations within the nerve. Brain Res. 2002;928(1–2):18‐29. https://doi.org10.1016/s0006-8993(01)03264-4

Leonard RB, Kevetter GA. Vestibular efferents contain peripherin. Neurosci Lett. 2006;408(2):104‐107. https://doi.org10.1016/j.neulet.2006.08.072

Oestreicher D, Picher MM, Rankovic V, Moser T, Pangrsic T. Cabp2‐gene therapy restores inner hair cell calcium currents and improves hearing in a DFNB93 mouse model. Front Mol Neurosci. 2021;14:689415.

Pangršič T, Gabrielaitis M, Michanski S, et al. EF‐hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells. Proc Natl Acad Sci USA. 2015;112(9):E1028‐E1037.

Leonard RB, Kevetter GA. Molecular probes of the vestibular nerve: I. Peripheral termination patterns of calretinin, calbindin and peripherin containing fibers. Brain Res. 2002;928(1–2):8‐17.

Prins TJ, Myers ZA, Saldate JJ, Hoffman LF. Calbindin expression in adult vestibular epithelia. J Comp Physiol A. 2020;206:623‐637.

Liu W, Davis RL. Calretinin and calbindin distribution patterns specify subpopulations of type I and type II spiral ganglion neurons in postnatal murine cochlea. J Comp Neurol. 2014;522(10):2299‐2318.

Desai SS, Ali H, Lysakowski A. Comparative morphology of rodent vestibular periphery. II Cristae Ampullares. J Neurophysiol. 2005;93(1):267‐280.

Dechesne CJ, Rabejac D, Desmadryl G. Development of calretinin immunoreactivity in the mouse inner ear. J Comp Neurol. 1994;346(4):517‐529.

Hackney CM, Mahendrasingam S, Penn A, Fettiplace R. The concentrations of calcium buffering proteins in mammalian cochlear hair cells. J Neurosci. 2005;25(34):7867‐7875.

Puyal J, Devau G, Venteo S, Sans N, Raymond J. Calcium‐binding proteins map the postnatal development of rat vestibular nuclei and their vestibular and cerebellar projections. J Comp Neurol. 2002;451(4):374‐391.

Desai SS, Zeh C, Lysakowski A. Comparative morphology of rodent vestibular periphery. I. Saccular and utricular maculae. J Neurophysiol. 2005;93(1):251‐266.

Eatock RA, Songer JE. Vestibular hair cells and afferents: two channels for head motion signals. Annu Rev Neurosci. 2011;34:501‐534.

Desmadryl G, Dechesne CJ. Calretinin immunoreactivity in chinchilla and Guinea pig vestibular end organs characterizes the calyx unit subpopulation. Exp Brain Res. 2004;89:105‐108.

Zheng JL, Gao WQ. Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker. J Neurosci. 1997;17(21):8270‐8282. https://doi.org10.1523/jneurosci.17-21-08270.1997

Kaiser M, Lüdtke TH, Deuper L, et al. TBX2 specifies and maintains inner hair and supporting cell fate in the organ of Corti. Nat Commun. 2022;13(1):7628.

Maklad A, Fritzsch B. The developmental segregation of posterior crista and saccular vestibular fibers in mice: a carbocyanine tracer study using confocal microscopy. Dev Brain Res. 2002;135(1–2):1‐17.

Maklad A, Fritzsch B. Incomplete segregation of endorgan‐specific vestibular ganglion cells in mice and rats. J Vestib Res. 1999;9(6):387‐399.

Maklad A, Kamel S, Wong E, Fritzsch B. Development and organization of polarity‐specific segregation of primary vestibular afferent fibers in mice. Cell Tissue Res. 2010;340:303‐321.

Maklad A, Fritzsch B. Partial segregation of posterior crista and saccular fibers to the nodulus and uvula of the cerebellum in mice, and its development. Dev Brain Res. 2003;140(2):223‐236.

Ji YR, Tona Y, Wafa T, et al. Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2. Nat Commun. 2022;13(1):6330.

Lysakowski A. Anatomy and Microstructural Organization of Vestibular Hair Cells. 2020.

Fernández C, Goldberg JM, Baird RA. The vestibular nerve of the chinchilla. III. Peripheral innervation patterns in the utricular macula. J Neurophysiol. 1990;63(4):767‐780. https://doi.org10.1152/jn.1990.63.4.767

Petitpré C, Faure L, Uhl P, et al. Single‐cell RNA‐sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification. Nat Commun. 2022;13(1):3878. https://doi.org10.1038/s41467-022-31580-1

Elliott KL, Kersigo J, Lee JH, et al. Developmental changes in peripherin‐eGFP expression in spiral ganglion neurons. Front Cell Neurosci. 2021;15:678113.

Glover JC, Fritzsch B. Molecular mechanisms governing development of the hindbrain choroid plexus and auditory projection: a validation of the seminal observations of Wilhelm his. IBRO Neurosci Rep. 2022;13:306‐313. https://doi.org10.1016/j.ibneur.2022.09.011

Chen Y, Takano‐Maruyama M, Fritzsch B, Gaufo GO. Hoxb1 controls anteroposterior identity of vestibular projection neurons. PLoS One. 2012;7(4):e34762. https://doi.org10.1371/journal.pone.0034762

Di Bonito M, Studer M. Cellular and molecular underpinnings of neuronal assembly in the central auditory system during mouse development. Front Neural Circuits. 2017;11:18.

Sun Y, Wang L, Zhu T, et al. Single‐cell transcriptomic landscapes of the otic neuronal lineage at multiple early embryonic ages. Cell Rep. 2022;38(12):110542.

Shrestha BR, Chia C, Wu L, Kujawa SG, Liberman MC, Goodrich LV. Sensory neuron diversity in the inner ear is shaped by activity. Cell. 2018;174(5):1229‐1246.e17. https://doi.org10.1016/j.cell.2018.07.007

Elliott KL, Iskusnykh IY, Chizhikov VV, Fritzsch B. Ptf1a expression is necessary for correct targeting of spiral ganglion neurons within the cochlear nuclei. Neurosci Lett. 2023;806:137244. https://doi.org10.1016/j.neulet.2023.137244

Butts JC, Wu SR, Durham MA, et al. A single‐cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain. Dev Cell. 2024;59:2171‐2188.e7. https://doi.org10.1016/j.devcel.2024.07.007

Jing J, Hu M, Ngodup T, et al. Molecular logic for cellular specializations that initiate the auditory parallel processing pathways. Nat Commun. 2025;16(1):489.

Puelles L, Nieuwenhuys R. Can we explain thousands of molecularly identified mouse neuronal types? From knowing to understanding. Biomolecules. 2024;14(6):708.

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(1):90‐109.

Fritzsch B, Dillard M, Lavado A, Harvey NL, Jahan I. Canal cristae growth and fiber extension to the outer hair cells of the mouse ear require Prox1 activity. PLoS One. 2010;5(2):e9377.

Matei V, Pauley S, Kaing S, et al. Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit. Dev Dyn. 2005;234(3):633‐650. https://doi.org10.1002/dvdy.20551

You D, Ni W, Huang Y, et al. The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function. Cell Mol Life Sci. 2023;80(12):349.

Burns JC, Stone JS. Development and regeneration of vestibular hair cells in mammals. Semin Cell Dev Biol. 2017;65:96‐105.

Wang T, Yang T, Kedaigle A, et al. Precise genetic control of ATOH1 enhances maturation of regenerated hair cells in the mature mouse utricle. Nat Commun. 2024;15(1):9166. https://doi.org10.1038/s41467-024-53153-0

Kopecky BJ, Jahan I, Fritzsch B. Correct timing of proliferation and differentiation is necessary for normal inner ear development and auditory hair cell viability. Dev Dyn. 2013;242(2):132‐147.

Jahan I, Pan N, Elliott KL, Fritzsch B. The quest for restoring hearing: understanding ear development more completely. BioEssays. 2015;37(9):1016‐1027.

Yamoah EN, Pavlinkova G, Fritzsch B. Molecular cascades that build and connect auditory neurons from hair cells to the auditory cortex. J Exp Neurol. 2025;6(3):111‐120.

Lysakowski A, Alonto A, Jacobson L. Peripherin immunoreactivity labels small diameter vestibular ‘bouton’ afferents in rodents. Hear Res. 1999;133(1–2):149‐154. https://doi.org10.1016/s0378-5955(99)00065-9

Simmons D, Duncan J, de Caprona DC, Fritzsch B. Development of the inner ear efferent system. Auditory and Vestibular efferents. Springer; 2010:187‐216.

Hafidi A. Peripherin‐like immunoreactivity in type II spiral ganglion cell body and projections. Brain Res. 1998;805(1–2):181‐190. https://doi.org10.1016/s0006-8993(98)00448-x

Ma Q, Anderson DJ, Fritzsch B. Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. J Assoc Res Otolaryngol. 2000;1:129‐143.

Yang T, Kersigo J, Jahan I, Pan N, Fritzsch B. The molecular basis of making spiral ganglion neurons and connecting them to hair cells of the organ of Corti. Hear Res. 2011;278(1–2):21‐33. https://doi.org10.1016/j.heares.2011.03.002

Filova I, Bohuslavova R, Tavakoli M, Yamoah EN, Fritzsch B, Pavlinkova G. Early deletion of Neurod1 alters neuronal lineage potential and diminishes neurogenesis in the inner ear. Front Cell Dev Biol. 2022;10:845461.

Johnson SB, Schmitz HM, Santi PA. TSLIM imaging and a morphometric analysis of the mouse spiral ganglion. Hear Res. 2011;278(1–2):34‐42.

Kopecky B, Santi P, Johnson S, Schmitz H, Fritzsch B. Conditional deletion of N‐Myc disrupts neurosensory and non‐sensory development of the ear. Dev Dyn. 2011;240(6):1373‐1390.

Buckiová D, Syka J. Calbindin and S100 protein expression in the developing inner ear in mice. J Comp Neurol. 2009;513(5):469‐482. https://doi.org10.1002/cne.21967

Lindeman HH. Studies on the morphology of the sensory regions of the vestibular apparatus with 45 figures. Ergeb Anat Entwicklungsgesch. 1969;42(1):1‐113.

Gianoli G, Goebel J, Mowry S, Poomipannit P. Anatomic differences in the lateral vestibular nerve channels and their implications in vestibular neuritis. Otol Neurotol. 2005;26(3):489‐494.

Rubio ME. Auditory brainstem development and plasticity. Curr Opin Physio. 2020;18:7‐10.

Yamoah EN, Pavlinkova G, Fritzsch B. The development of speaking and singing in infants may play a role in genomics and dementia in humans. Brain Sci. 2023;13(8):1190.

Harris L, Rigo P, Stiehl T, et al. Coordinated changes in cellular behavior ensure the lifelong maintenance of the hippocampal stem cell population. Cell Stem Cell. 2021;28(5):863‐876.e6. https://doi.org10.1016/j.stem.2021.01.003

Ono K, Keller J, López Ramírez O, et al. Retinoic acid degradation shapes zonal development of vestibular organs and sensitivity to transient linear accelerations. Nat Commun. 2020;11(1):63.

Simmons DD, Tong B, Schrader AD, Hornak AJ. Oncomodulin identifies different hair cell types in the mammalian inner ear. J Comp Neurol. 2010;518(18):3785‐3802.

Sanders TR, Kelley MW. Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse. Proc Natl Acad Sci USA. 2022;119(48):e2203935119.

Siebald C, Vincent PF, Bottom RT, et al. Molecular signatures define subtypes of auditory afferents with distinct peripheral projection patterns and physiological properties. Proc Natl Acad Sci USA. 2023;120(31):e2217033120.

Fritzsch B, Weng X, Yamoah EN, et al. Irx3/5 null deletion in mice blocks cochlea‐saccule segregation and disrupts the auditory tonotopic map. J Comp Neurol. 2024;532:e70008 https://doi.org10.1002/cne.70008

Elliott KL, Kersigo J, Lee JH, Yamoah EN, Fritzsch B. Sustained loss of Bdnf affects peripheral but not central vestibular targets. Front Neurol. 2021;12:768456.

Malmierca MS. Auditory system. The rat nervous system. Elsevier; 2015:865‐946.

Vidal P–P, Cullen K, Curthoys I, et al. The vestibular system. The Rat Nervous system. Elsevier; 2015:805‐864.

McLenachan S, Goldshmit Y, Fowler KJ, et al. Transgenic mice expressing the peripherin‐EGFP genomic reporter display intrinsic peripheral nervous system fluorescence. Transgenic Res. 2008;17(6):1103‐1116. https://doi.org10.1007/s11248-008-9210-7

Johnson KR, Zheng QY, Erway LC. A major gene affecting age‐related hearing loss is common to at least ten inbred strains of mice. Genomics. 2000;70(2):171‐180.

Erway LC, Shiau Y‐W, Davis RR, Krieg EF. Genetics of age‐related hearing loss in mice. III. Susceptibility of inbred and F1 hybrid strains to noise‐induced hearing loss. Hear Res. 1996;93(1–2):181‐187.

Elliott KL, Fritzsch B, Yamoah EN, Zine A. Age‐related hearing loss: sensory and neural etiology and their interdependence. Front Aging Neurosci. 2022;14:814528.

Schmidt H, Fritzsch B. Npr2 null mutants show initial overshooting followed by reduction of spiral ganglion axon projections combined with near‐normal cochleotopic projection. Cell Tissue Res. 2019;378:15‐32.

Dicer Deletion in the Ear Can Cut Most Neurons and Their Innervation of Hair Cells to Project to the Ear and the Brainstem

Regulatory Networks Driving the Specification, Differentiation, and Diversification of Neurons in the Mouse Inner Ear