A gene cadre orchestrates the normal development of sensory and non-sensory cells in the inner ear, segregating the cochlea with a distinct tonotopic sound frequency map, similar brain projection, and five vestibular end-organs. However, the role of genes driving the ear development is largely unknown. Here, we show double deletion of the Iroquois homeobox 3 and 5 transcription factors (Irx3/5 DKO) leads to the fusion of the saccule and the cochlear base. The overlying otoconia and tectorial membranes are absent in the Irx3/5 DKO inner ear, and the primary auditory neurons project fibers to both the saccule and cochlear hair cells. The central neuronal projections from the cochlear apex-base contour are not fully segregated into a dorsal and ventral innervation in the Irx3/5 DKO cochlear nucleus, obliterating the characteristic tonotopic auditory map. Additionally, Irx3/5 deletion reveals a pronounced cochlear-apex-vestibular "vestibular-cochlear" nerve (VCN) bilateral connection that is less noticeable in wild-type control mice. Moreover, the incomplete segregation of apex and base projections that expands fibers to connect with vestibular nuclei. The results suggest the mammalian cochlear apex is a derived lagena reminiscent of sarcopterygians. Thus, Irx3 and 5 are potential evolutionary branch-point genes necessary for balance-sound segregation, which fused into a saccule-cochlea organization.

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

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

Laboratory of Molecular Pathogenetics Institute of Biotechnology CAS Vestec Czechia

Program in Developmental and Stem Cell Biology The Hospital for Sick Children Toronto Ontario Canada

School of Biomedical Sciences The Chinese University of Hong Kong Shatin Hong Kong SAR China

Zobrazit více v PubMed

Bi, Z. , Ren M., Zhang Y., et al. 2024. “Revisiting the Potency of Tbx2 Expression in Transforming Outer Hair Cells Into Inner Hair Cells at Multiple Ages In Vivo.” Journal of Neuroscience 44, no. 23: e1751232024. PubMed PMC

Bok, J. , Zenczak C., Hwang C. H., and Wu D. K.. 2013. “Auditory Ganglion Source of Sonic Hedgehog Regulates Timing of Cell Cycle Exit and Differentiation of Mammalian Cochlear Hair Cells.” Proceedings of the National Academy of Sciences of the United States of America 110: 13869–13874. PubMed PMC

Bouchard, M. , de Caprona D., Busslinger M., Xu P., and Fritzsch B.. 2010. “Pax2 and Pax8 Cooperate in Mouse Inner Ear Morphogenesis and Innervation.” BMC Developmental Biology 10: 1–17. PubMed PMC

Burton, Q. , Cole L. K., Mulheisen M., Chang W., and Wu D. K.. 2004. “The Role of Pax2 in Mouse Inner Ear Development.” Developmental Biology 272: 161–175. PubMed

Cain, C. J. , Gaborit N., Lwin W., et al. 2016. “Loss of Iroquois Homeobox Transcription Factors 3 and 5 in Osteoblasts Disrupts Cranial Mineralization.” Bone Reports 5: 86–95. PubMed PMC

Cardeña‐Núñez, S. , Sánchez‐Guardado L. O., Corral‐San‐Miguel R., et al. 2017. “Expression Patterns of Irx Genes in the Developing Chick Inner Ear.” Brain Structure and Function 222: 2071–2092. PubMed

Chizhikov, V. V. , Iskusnykh I. Y., Fattakhov N., and Fritzsch B.. 2021. “Lmx1a and Lmx1b Are Redundantly Required for the Development of Multiple Components of the Mammalian Auditory System.” Neuroscience 452: 247–264. PubMed PMC

de Sousa Lobo Ferreira Querido, R. , Ji X., Lakha R., et al. 2023. “Visualizing Collagen Fibrils in the Cochlea's Tectorial and Basilar Membranes Using a Fluorescently Labeled Collagen‐Binding Protein Fragment.” Journal of the Association for Research in Otolaryngology 24: 147–157. PubMed PMC

Dou, Z. , Son J. E., and Hui C.‐C.. 2021. “Irx3 and Irx5‐Novel Regulatory Factors of Postnatal Hypothalamic Neurogenesis.” Frontiers in Neuroscience 15: 763856. PubMed PMC

Driver, E. C. , and Kelley M. W.. 2020. “Development of the Cochlea.” Development 147, no. 12: dev162263. PubMed PMC

Duncan, J. S. , and Fritzsch B.. 2013. “Continued Expression of GATA3 Is Necessary for Cochlear Neurosensory Development.” PLoS ONE 8: e62046. PubMed PMC

Dvorakova, M. , Macova I., Bohuslavova R., Anderova M., Fritzsch B., and Pavlinkova G.. 2020. “Early Ear Neuronal Development, but Not Olfactory or Lens Development, Can Proceed Without SOX2.” Developmental Biology 457: 43–56. PubMed PMC

Elliott, K. L. , Iskusnykh I. Y., Chizhikov V. V., and Fritzsch B.. 2023. “Ptf1a Expression Is Necessary for Correct Targeting of Spiral Ganglion Neurons Within the Cochlear Nuclei.” Neuroscience Letters 806: 137244. PubMed PMC

Elliott, K. L. , Pavlínková G., Chizhikov V. V., Yamoah E. N., and Fritzsch B.. 2021. “Development in the Mammalian Auditory System Depends on Transcription Factors.” International Journal of Molecular Sciences 22: 4189. PubMed PMC

Exelby, K. , Herrera‐Delgado E., Perez L. G., et al. 2021. “Precision of Tissue Patterning Is Controlled by Dynamical Properties of Gene Regulatory Networks.” Development 148: dev197566. PubMed PMC

Farmer, D. J. T. , Patel P., Choi R., Liu C.‐Y., and Crump J. G.. 2021. “A Comprehensive Series of Irx Cluster Mutants Reveals Diverse Roles in Facial Cartilage Development.” Development 148: dev197244. PubMed PMC

Filova, I. , Bohuslavova R., Tavakoli M., Yamoah E. N., Fritzsch B., and Pavlinkova G.. 2022. “Early Deletion of Neurod1 Alters Neuronal Lineage Potential and Diminishes Neurogenesis in the Inner Ear.” Frontiers in Cell and Developmental Biology 10: 845461–845461. PubMed PMC

Filova, I. , Pysanenko K., Tavakoli M., et al. 2022. “ISL1 Is Necessary for Auditory Neuron Development and Contributes Toward Tonotopic Organization.” Proceedings of the National Academy of Sciences of the United States of America 119: e2207433119. PubMed PMC

Fritzsch, B. , Dillard M., Lavado A., Harvey N. L., and Jahan I.. 2010. “Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity.” PLoS ONE 5: e9377. PubMed PMC

Fritzsch, B. , and Elliott K. L.. 2023. “Fish Hearing Revealed: Do We Understand Hearing in Critical Fishes and Marine Tetrapods.” Journal of the Acoustical Society of America 154: 3019–3026. PubMed PMC

Fritzsch, B. , Pan N., Jahan I., et al. 2013. “Evolution and Development of the Tetrapod Auditory System: An Organ of Corti‐Centric Perspective.” Evolution & Development 15: 63–79. PubMed PMC

Fritzsch, B. , Schultze H.‐P., and Elliott K. L.. 2023. “The Evolution of the Various Structures Required for Hearing in Latimeria and Tetrapods.” IBRO Neuroscience Reports 14: 325–341. PubMed PMC

Goodyear, R. J. , Lu X., Deans M. R., and Richardson G. P.. 2017. “A Tectorin‐Based Matrix and Planar Cell Polarity Genes Are Required for Normal Collagen‐Fibril Orientation in the Developing Tectorial Membrane.” Development 144: 3978–3989. PubMed PMC

Goodyear, R. J. , and Richardson G. P.. 2018. “Structure, Function, and Development of the Tectorial Membrane: An Extracellular Matrix Essential for Hearing.” Current Topics in Developmental Biology 130: 217–244. PubMed

Jahan, I. , Pan N., Kersigo J., and Fritzsch B.. 2015. “Neurog1 Can Partially Substitute for Atoh1 Function in Hair Cell Differentiation and Maintenance During Organ of Corti Development.” Development 142: 2810–2821. PubMed PMC

Jean, P. , Wong Jun Tai F., Singh‐Estivalet A., et al. 2023. “Single‐Cell Transcriptomic Profiling of the Mouse Cochlea: An Atlas for Targeted Therapies.” Proceedings of the National Academy of Sciences of the United States of America 120: e2221744120. PubMed PMC

Jiang, B. , Huang L., Tian T., et al. 2022. “IRX5 Promotes Adipogenesis of hMSCs by Repressing Glycolysis.” Cell Death Discovery 8: 204. PubMed PMC

Kaiser, M. , Lüdtke T. H., Deuper L., et al. 2022. “TBX2 Specifies and Maintains Inner Hair and Supporting Cell Fate in the Organ of Corti.” Nature Communications 13: 7628. PubMed PMC

Kerner, P. , Ikmi A., Coen D., and Vervoort M.. 2009. “Evolutionary History of the Iroquois/Irx Genes in Metazoans.” BMC Evolutionary Biology 9: 1–14. PubMed PMC

Koh, J.‐Y. , Affortit C., Ranum P. T., et al. 2023. “Single‐Cell RNA‐Sequencing of Stria Vascularis Cells in the Adult Slc26a4‐/‐Mouse.” BMC Medical Genomics 16: 1–17. PubMed PMC

Koo, S. K. , Hill J. K., Hwang C. H., Lin Z. S., Millen K. J., and Wu D. K.. 2009. “Lmx1a Maintains Proper Neurogenic, Sensory, and Non‐Sensory Domains in the Mammalian Inner Ear.” Developmental Biology 333: 14–25. PubMed PMC

Kopecky, B. , Johnson S., Schmitz H., Santi P., and Fritzsch B.. 2012. “Scanning Thin‐Sheet Laser Imaging Microscopy Elucidates Details on Mouse Ear Development.” Developmental Dynamics 241: 465–480. PubMed PMC

Kopecky, B. , Santi P., Johnson S., Schmitz H., and Fritzsch B.. 2011. “Conditional Deletion of N‐Myc Disrupts Neurosensory and Non‐Sensory Development of the Ear.” Developmental Dynamics 240: 1373–1390. PubMed PMC

Leung, B.‐W. , Liu Y., Qin T., So K., Hui C., and Sham M.. 2019. “The Roles of Irx3 and Irx5 in Cochlear Epithelial Cell Fate Specification.” Paper presented at the European Developmental Biology Congress (EDBC), Alicante, Spain, October 23–26, 2019.

Li, D. , Sakuma R., Vakili N. A., et al. 2014. “Formation of Proximal and Anterior Limb Skeleton Requires Early Function of Irx3 and Irx5 and Is Negatively Regulated by Shh Signaling.” Developmental Cell 29: 233–240. PubMed

Liu, Y. , Qin T., Weng X., et al. 2024. “Irx3/5 Define the Cochlear Sensory Domain and Regulate Vestibular and Cochlear Sensory Patterning in the Mammalian Inner Ear.” bioRxiv.

Liu, Y. , Wang B., Wong E. Y. M., et al. 2017. “The Roles of Irx3 and Irx5 in Inner Ear Neurosensory Patterning.” Mechanisms of Development 145: S120–S121.

Lorenzen, S. M. , Duggan A., Osipovich A. B., Magnuson M. A., and García‐Añoveros J.. 2015. “Insm1 Promotes Neurogenic Proliferation in Delaminated Otic Progenitors.” Mechanisms of Development 138: 233–245. PubMed PMC

Luo, Z. , Du Y., Li S., et al. 2022. “Three Distinct Atoh1 Enhancers Cooperate for Sound Receptor Hair Cell Development.” Proceedings of the National Academy of Sciences of the United States of America 119: e2119850119. PubMed PMC

Luo, Z. , Zhang J., Qiao L., Lu F., and Liu Z.. 2021. “Mapping Genome‐Wide Binding Sites of Prox1 in Mouse Cochlea Using the CUT&RUN Approach.” Neuroscience Bulletin 37: 1703–1707. PubMed PMC

Macova, I. , Pysanenko K., Chumak T., et al. 2019. “Neurod1 Is Essential for the Primary Tonotopic Organization and Related Auditory Information Processing in the Midbrain.” Journal of Neuroscience 39: 984–1004. PubMed PMC

Maklad, A. , and Fritzsch B.. 2002. “The Developmental Segregation of Posterior Crista and Saccular Vestibular Fibers in Mice: A Carbocyanine Tracer Study Using Confocal Microscopy.” Developmental Brain Research 135: 1–17. PubMed

Manley, G. A. 2023. “Evolution of Diversity in the Auditory Papillae of Reptiles.” Diversity 15: 730.

Mann, Z. F. , Gálvez H., Pedreno D., et al. 2017. “Shaping of Inner Ear Sensory Organs Through Antagonistic Interactions Between Notch Signalling and Lmx1a.” Elife 6: e33323. PubMed PMC

Marlétaz, F. , Timoshevskaya N., Timoshevskiy V. A., et al. 2024. “The Hagfish Genome and the Evolution of Vertebrates.” Nature 627, no. 8005: 811–820. PubMed PMC

Matei, V. , Pauley S., Kaing S., et al. 2005. “Smaller Inner Ear Sensory Epithelia in Neurog1 Null Mice Are Related to Earlier Hair Cell Cycle Exit.” Developmental Dynamics 234: 633–650. PubMed PMC

Mulry, E. , and Parham K.. 2020. “Inner Ear Proteins as Potential Biomarkers.” Otology & Neurotology 41: 145–152. PubMed

Muthu, V. , Rohacek A. M., Yao Y., et al. 2019. “Genomic Architecture of Shh‐Dependent Cochlear Morphogenesis.” Development 146: dev181339. PubMed PMC

Narwidina, A. , Miyazaki A., Iwata K., et al. 2023. “Iroquois Homeobox 3 Regulates Odontoblast Proliferation and Differentiation Mediated by Wnt5a Expression.” Biochemical and Biophysical Research Communications 650: 47–54. PubMed

Newton, A. H. , Williams S. M., Major A. T., and Smith C. A.. 2022. “Cell Lineage Specification and Signalling Pathway Use During Development of the Lateral Plate Mesoderm and Forelimb Mesenchyme.” Development 149, no. 18: dev200702. PubMed

Nichols, D. H. , Pauley S., Jahan I., Beisel K. W., Millen K. J., and Fritzsch B.. 2008. “Lmx1a Is Required for Segregation of Sensory Epithelia and Normal Ear Histogenesis and Morphogenesis.” Cell and Tissue Research 334: 339–358. PubMed PMC

Pauley, S. , Lai E., and Fritzsch B.. 2006. “Foxg1 Is Required for Morphogenesis and Histogenesis of the Mammalian Inner Ear.” Developmental Dynamics 235: 2470–2482. PubMed PMC

Pressé, M. T. , Malgrange B., and Delacroix L.. 2023. “The Cochlear Matrisome: Importance in Hearing and Deafness.” Matrix Biology 125: 40–58. PubMed

Pyott, S. J. , Pavlinkova G., Yamoah E. N., and Fritzsch B.. 2024. “Harmony in the Molecular Orchestra of Hearing: Developmental Mechanisms From the Ear to the Brain.” Annual Review of Neuroscience 47: 1–20. PubMed PMC

Schmidt, H. , and Fritzsch B.. 2019. “Npr2 Null Mutants Show Initial Overshooting Followed by Reduction of Spiral Ganglion Axon Projections Combined With Near‐Normal Cochleotopic Projection.” Cell and Tissue Research 378: 15–32. PubMed PMC

Schultz, J. A. , Zeller U., and Luo Z. X.. 2017. “Inner Ear Labyrinth Anatomy of Monotremes and Implications for Mammalian Inner Ear Evolution.” Journal of Morphology 278: 236–263. PubMed

Steffes, G. , Lorente‐Cánovas B., Pearson S., et al. 2012. “Mutanlallemand (mtl) and Belly Spot and Deafness (BSD) Are Two New Mutations of Lmx1a Causing Severe Cochlear and Vestibular Defects.” PLoS ONE 7: e51065. PubMed PMC

Sun, Y. , and Liu Z.. 2023. “Recent Advances in Molecular Studies on Cochlear Development and Regeneration.” Current Opinion in Neurobiology 81: 102745. PubMed

Susaki, E. A. , Tainaka K., Perrin D., Yukinaga H., Kuno A., and Ueda H. R.. 2015. “Advanced CUBIC Protocols for Whole‐Brain and Whole‐Body Clearing and Imaging.” Nature Protocols 10: 1709–1727. PubMed

Tan, Z. , Kong M., Wen S., et al. 2020. “IRX3 and IRX5 Inhibit Adipogenic Differentiation of Hypertrophic Chondrocytes and Promote Osteogenesis.” Journal of Bone and Mineral Research 35: 2444–2457. PubMed

Tao, H. , Lambert J. P., Yung T. M., et al. 2020. “IRX3/5 Regulate Mitotic Chromatid Segregation and Limb Bud Shape.” Development 147, no. 19: dev180042. PubMed PMC

Wang, G. , Gu Y., and Liu Z.. 2024. “Deciphering the Genetic Interactions Between Pou4f3, Gfi1, and Rbm24 in Maintaining Mouse Cochlear Hair Cell Survival.” Elife 12: RP90025. PubMed PMC

Wu, J. , Zhang Y., Mao S., et al. 2023. “Cross‐Species Analysis and Comparison of the Inner Ear Between Chickens and Mice.” Journal of Comparative Neurology 531: 1443–1458. PubMed

Xu, J. , Li J., Zhang T., et al. 2021. “Chromatin Remodelers and Lineage‐Specific Factors Interact to Target Enhancers to Establish Proneurosensory Fate Within Otic Ectoderm.” Proceedings of the National Academy of Sciences of the United States of America 118: e2025196118. PubMed PMC

Yu, D. , Ren Y., Uesaka M., et al. 2024. “Hagfish Genome Elucidates Vertebrate Whole‐Genome Duplication Events and Their Evolutionary Consequences.” Nature Ecology & Evolution 8: 519–535. PubMed PMC

Zheng, J. L. , and Gao W.‐Q.. 2000. “Overexpression of Math1 Induces Robust Production of Extra Hair Cells in Postnatal Rat Inner Ears.” Nature Neuroscience 3: 580–586. PubMed