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Combined Atoh1 and Neurod1 Deletion Reveals Autonomous Growth of Auditory Nerve Fibers

Abstract

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.

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Acknowledgments

We thank O. Benada of the Electron Microscopy Group (Institute of Microbiology CAS) for assistance with SEM and acknowledge the support of the Imaging Methods Core Facility at BIOCEV funded by the Czech-BioImaging large RI projects (LM2015062 and CZ.02.1.01/0.0/0.0/16_013/0001775, from MEYS CR).

Funding

This study was supported by a grant from the Czech Science Foundation (20-06927S to GP); by BIOCEVCZ.1.05/1.1.00/02.0109 from the ERDF; by the institutional support of the Czech Academy of Sciences RVO: 86652036; and by NIH (R01 AG060504 to BF).

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Contributions

G.P. and B.F. conceptualized the work and wrote the MS. I.M. wrote the first draft of the paper. I.M., M.D., S.V., K.L.E., and R.B. performed in situ hybridization and immunohistological analyses. R.B. prepared and validated RNA for RNA-Seq. A.P. performed qPCR and RNA-Seq bioinformatics analyses and the editing of the MS. B.F. performed dye tracing.

Corresponding authors

Correspondence to Bernd Fritzsch or Gabriela Pavlinkova.

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The authors declare that they have no conflict of interest.

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All methods were performed in agreement with the Guide for the Care and Use of Laboratory Animals (National Research Council. Washington, DC. The National Academies Press, 1996). The design of experiments was approved by the Animal Care and Use Committee of the Institute of Molecular Genetics, Czechia.

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Filova, I., Dvorakova, M., Bohuslavova, R. et al. Combined Atoh1 and Neurod1 Deletion Reveals Autonomous Growth of Auditory Nerve Fibers. Mol Neurobiol 57, 5307–5323 (2020). https://doi.org/10.1007/s12035-020-02092-0

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Keywords

  • bHLH genes
  • Ear neurosensory development
  • Neuronal differentiation
  • Central projections
  • Axon guidance