Ear development requires the transcription factors ATOH1 for hair cell differentiation and NEUROD1 for sensory neuron development. In addition, NEUROD1 negatively regulates Atoh1 gene expression. As we previously showed that deletion of the Neurod1 gene in the cochlea results in axon guidance defects and excessive peripheral innervation of the sensory epithelium, we hypothesized that some of the innervation defects may be a result of abnormalities in NEUROD1 and ATOH1 interactions. To characterize the interdependency of ATOH1 and NEUROD1 in inner ear development, we generated a new Atoh1/Neurod1 double null conditional deletion mutant. Through careful comparison of the effects of single Atoh1 or Neurod1 gene deletion with combined double Atoh1 and Neurod1 deletion, we demonstrate that NEUROD1-ATOH1 interactions are not important for the Neurod1 null innervation phenotype. We report that neurons lacking Neurod1 can innervate the flat epithelium without any sensory hair cells or supporting cells left after Atoh1 deletion, indicating that neurons with Neurod1 deletion do not require the presence of hair cells for axon growth. Moreover, transcriptome analysis identified genes encoding axon guidance and neurite growth molecules that are dysregulated in the Neurod1 deletion mutant. Taken together, we demonstrate that much of the projections of NEUROD1-deprived inner ear sensory neurons are regulated cell-autonomously.

Department of Biology University of Iowa Iowa City IA 52242 1324 USA

Institute of Biotechnology of the Czech Academy of Sciences 25250 Vestec Czechia

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Driver EC, Kelley MW (2020) Development of the cochlea. Development 147 (12). doi:10.1242/dev.162263 PubMed DOI PMC

Zou D, Silvius D, Fritzsch B, Xu P-X (2004) Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes. Development 131 (22):5561–5572 PubMed PMC

Kiernan AE, Pelling AL, Leung KK, Tang AS, Bell DM, Tease C, Lovell-Badge R, Steel KP, Cheah KS (2005) Sox2 is required for sensory organ development in the mammalian inner ear. Nature 434 (7036):1031–1035 PubMed

Karis A, Pata I, van Doorninck JH, Grosveld F, de Zeeuw CI, de Caprona D, Fritzsch B (2001) Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear. J Comp Neurol 429 (4):615–630 PubMed

Bouchard M, de Caprona D, Busslinger M, Xu P, Fritzsch B (2010) Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation. BMC developmental biology 10 (1):89. PubMed PMC

Raft S, Groves AK (2014) Segregating neural and mechanosensory fates in the developing ear: patterning, signaling, and transcriptional control. Cell and tissue research. doi:10.1007/s00441-014-1917-6 PubMed DOI PMC

Fritzsch B, Elliott KL (2017) Gene, cell, and organ multiplication drives inner ear evolution. Developmental Biology 431 (1):3–15. doi:10.1016/j.ydbio.2017.08.034 PubMed DOI PMC

Ma Q, Anderson DJ, Fritzsch B (2000) Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. Journal of the Association for Research in Otolaryngology 1 (2):129–143 PubMed PMC

Kim W-Y, Fritzsch B, Serls A, Bakel LA, Huang EJ, Reichardt LF, Barth DS, Lee JE (2001) NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development 128 (3):417–426 PubMed PMC

Jahan I, Pan N, Kersigo J, Fritzsch B (2010) Neurod1 suppresses hair cell differentiation in ear ganglia and regulates hair cell subtype development in the cochlea. PloS one 5 (7):e11661. doi:10.1371/journal.pone.0011661 PubMed DOI PMC

Kopecky BJ, Jahan I, Fritzsch B (2013) Correct timing of proliferation and differentiation is necessary for normal inner ear development and auditory hair cell viability. Developmental Dynamics 242 (2):132–147 PubMed PMC

Pan N, Jahan I, Kersigo J, Duncan JS, Kopecky B, Fritzsch B (2012) A novel Atoh1 “self-terminating” mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability. PloS one 7 (1):e30358. doi:10.1371/journal.pone.0030358 PubMed DOI PMC

Pan N, Jahan I, Kersigo J, Kopecky B, Santi P, Johnson S, Schmitz H, Fritzsch B (2011) Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti. Hearing research 275 (1–2):66–80. doi:10.1016/j.heares.2010.12.002 PubMed DOI PMC

Bermingham N, Hassan B, Price S, Vollrath M, Ben-Arie N, Eatock R, Bellen H, Lysakowski A, Zoghbi H (1999) Math1: an essential gene for the generation of inner ear hair cells. Science (284):1837–1841 PubMed

Macova I, Pysanenko K, Chumak T, Dvorakova M, Bohuslavova R, Syka J, Fritzsch B, Pavlinkova G (2019) Neurod1 Is Essential for the Primary Tonotopic Organization and Related Auditory Information Processing in the Midbrain. J Neurosci 39 (6):984–1004. doi:10.1523/JNEUROSCI.2557-18.2018 PubMed DOI PMC

Yang L, Cai CL, Lin L, Qyang Y, Chung C, Monteiro RM, Mummery CL, Fishman GI, Cogen A, Evans S (2006) Isl1Cre reveals a common Bmp pathway in heart and limb development. Development 133 (8):1575–1585. doi:10.1242/dev.02322 PubMed DOI PMC

Goebbels S, Bode U, Pieper A, Funfschilling U, Schwab MH, Nave KA (2005) Cre/loxP-mediated inactivation of the bHLH transcription factor gene NeuroD/BETA2. Genesis 42 (4):247–252. doi:10.1002/gene.20138 PubMed DOI

Shroyer NF, Helmrath MA, Wang VY, Antalffy B, Henning SJ, Zoghbi HY (2007) Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis. Gastroenterology 132 (7):2478–2488. doi:10.1053/j.gastro.2007.03.047 PubMed DOI

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 6:38253. doi:10.1038/srep38253 PubMed DOI PMC

Bohuslavova R, Dodd N, Macova I, Chumak T, Horak M, Syka J, Fritzsch B, Pavlinkova G (2017) Pax2-Islet1 Transgenic Mice Are Hyperactive and Have Altered Cerebellar Foliation. Mol Neurobiol 54 (2):1352–1368. doi:10.1007/s12035-016-9716-6 PubMed DOI PMC

Fritzsch B, Duncan JS, Kersigo J, Gray B, Elliott KL (2016) Neuroanatomical Tracing Techniques in the Ear: History, State of the Art, and Future Developments In: Sokolowski B, Ed: Auditory and Vestibular Research: Methods and Protocols, vol 1427 Springer Science+Business Media New York, pp 243–262 PubMed PMC

Simmons D, Duncan J, de Caprona DC, Fritzsch B (2011) Development of the inner ear efferent system In: Auditory and vestibular efferents. Springer, pp 187–216

Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 (15):2114–2120. doi:10.1093/bioinformatics/btu170 PubMed DOI PMC

Kopylova E, Noe L, Touzet H (2012) SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28 (24):3211–3217. doi:10.1093/bioinformatics/bts611 PubMed DOI

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29 (1):15–21. doi:10.1093/bioinformatics/bts635 PubMed DOI PMC

Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31 (2):166–169. doi:10.1093/bioinformatics/btu638 PubMed DOI PMC

Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15 (12):550. doi:10.1186/s13059-014-0550-8 PubMed DOI PMC

Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4 (1):44–57. doi:10.1038/nprot.2008.211 PubMed DOI

Fritzsch B, Matei VA, Nichols DH, Bermingham N, Jones K, Beisel KW, Wang VY (2005) Atoh1 null mice show directed afferent fiber growth to undifferentiated ear sensory epithelia followed by incomplete fiber retention. Dev Dyn 233 (2):570–583 PubMed PMC

Liu M, Pereira FA, Price SD, Chu M-j, Shope C, Himes D, Eatock RA, Brownell WE, Lysakowski A, Tsai M-J (2000) Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes & development 14 (22):2839–2854 PubMed PMC

Tateya T, Sakamoto S, Ishidate F, Hirashima T, Imayoshi I, Kageyama R (2019) Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea. Development 146 (21). doi:10.1242/dev.177881 PubMed DOI

Elliott KL, Kersigo J, Pan N, Jahan I, Fritzsch B (2017) Spiral ganglion neuron projection development to the hindbrain in mice lacking peripheral and/or central target differentiation. Frontiers in neural circuits 11:25. PubMed PMC

Yang T, Kersigo J, Jahan I, Pan N, Fritzsch B (2011) The molecular basis of making spiral ganglion neurons and connecting them to hair cells of the organ of Corti. Hearing research 278 (1–2):21–33 PubMed PMC

Lu CC, Appler JM, Houseman EA, Goodrich LV (2011) Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly. J Neurosci 31 (30):10903–10918. doi:10.1523/JNEUROSCI.2358-11.2011 PubMed DOI PMC

Fariñas I, Jones KR, Tessarollo L, Vigers AJ, Huang E, Kirstein M, De Caprona DC, Coppola V, Backus C, Reichardt LF (2001) Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression. Journal of Neuroscience 21 (16):6170–6180 PubMed PMC

Shi Y, Chichung Lie D, Taupin P, Nakashima K, Ray J, Yu RT, Gage FH, Evans RM (2004) Expression and function of orphan nuclear receptor TLX in adult neural stem cells. Nature 427 (6969):78–83. doi:10.1038/nature02211 PubMed DOI

Osterhout JA, Stafford BK, Nguyen PL, Yoshihara Y, Huberman AD (2015) Contactin-4 mediates axon-target specificity and functional development of the accessory optic system. Neuron 86 (4):985–999. doi:10.1016/j.neuron.2015.04.005 PubMed DOI PMC

Mercati O, Danckaert A, Andre-Leroux G, Bellinzoni M, Gouder L, Watanabe K, Shimoda Y, Grailhe R, De Chaumont F, Bourgeron T, Cloez-Tayarani I (2013) Contactin 4, −5 and −6 differentially regulate neuritogenesis while they display identical PTPRG binding sites. Biol Open 2 (3):324–334. doi:10.1242/bio.20133343 PubMed DOI PMC

Shibata SB, Budenz CL, Bowling SA, Pfingst BE, Raphael Y (2011) Nerve maintenance and regeneration in the damaged cochlea. Hearing research 281 (1–2):56–64. doi:10.1016/j.heares.2011.04.019 PubMed DOI PMC

Huang EJ, Liu W, Fritzsch B, Bianchi LM, Reichardt LF, Xiang M (2001) Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons. Development 128 (13):2421–2432 PubMed PMC

Fritzsch B, Barbacid M, Silos-Santiago I (1998) The combined effects of trkB and trkC mutations on the innervation of the inner ear. Int J Dev Neurosci 16 (6):493–505 PubMed

Deng M, Yang H, Xie X, Liang G, Gan L (2014) Comparative expression analysis of POU4F1, POU4F2 and ISL1 in developing mouse cochleovestibular ganglion neurons. Gene Expr Patterns 15 (1):31–37. doi:10.1016/j.gep.2014.03.001 PubMed DOI PMC

Lorenzen SM, Duggan A, Osipovich AB, Magnuson MA, Garcia-Anoveros J (2015) Insm1 promotes neurogenic proliferation in delaminated otic progenitors. Mech Dev 138 Pt 3:233–245. doi:10.1016/j.mod.2015.11.001 PubMed DOI PMC

Wiwatpanit T, Lorenzen SM, Cantú JA, Foo CZ, Hogan AK, Márquez F, Clancy JC, Schipma MJ, Cheatham MA, Duggan A (2018) Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1. Nature:1 PubMed PMC

Cramer KS (2005) Eph proteins and the assembly of auditory circuits. Hearing research 206 (1–2):42–51. doi:10.1016/j.heares.2004.11.024 PubMed DOI

Frisina RD, Ding B, Zhu X, Walton JP (2016) Age-related hearing loss: prevention of threshold declines, cell loss and apoptosis in spiral ganglion neurons. Aging (Albany NY) 8 (9):2081. PubMed PMC

Sha S-H, Schacht J (2017) Emerging therapeutic interventions against noise-induced hearing loss. Expert opinion on investigational drugs 26 (1):85–96 PubMed PMC

Fernandez KA, Jeffers PW, Lall K, Liberman MC, Kujawa SG (2015) Aging after noise exposure: acceleration of cochlear synaptopathy in “recovered” ears. J Neurosci 35 (19):7509–7520. doi:10.1523/JNEUROSCI.5138-14.2015 PubMed DOI PMC

Abbas PJ, Tejani VD, Scheperle RA, Brown CJ (2017) Using neural response telemetry to monitor physiological responses to acoustic stimulation in hybrid cochlear implant users. Ear and hearing 38 (4):409–425 PubMed PMC

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