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The mammalian cochlea is composed of sensory hair cells as well as multiple different types of non-sensory supporting cells. Pillar cells are one type of supporting cell that form the tunnel of Corti and include two morphologically and functionally distinct subtypes: inner pillar cells (IPCs) and outer pillar cells (OPCs). The processes of specification and differentiation of inner versus outer pillar cells are still unclear. Here, we show that β-Catenin is required for establishing IPC identity in the mammalian cochlea. To differentiate the transcriptional and adhesion roles of β-Catenin in establishing IPC identity, we examined two different models of β-Catenin deletion; one that deletes both transcriptional and structural functions and one which retains cell adhesion function but lacks transcriptional function. Here, we show that cochleae lacking β-Catenin transcriptional function lost IPCs and displayed extranumerary OPCs, indicating its requirement for establishing IPC identity. Overexpression of β-Catenin induced proliferation within IPCs but not ectopic IPCs. Single-cell transcriptomes of supporting cells lacking β-Catenin transcriptional function show a loss of the IPC and gain of OPC signatures. Finally, targeted deletion of β-Catenin in IPCs also led to the loss of IPC identity, indicating a cell autonomous role of β-Catenin in establishing IPC identity. As IPCs have the capacity to regenerate sensory hair cells in the postnatal cochlea, our results will aid in future IPC-based hair cell regeneration strategies. Author summary: The organ of Corti, the hearing apparatus, requires a precise arrangement of various cell types including hair cells (required for sound reception) and supporting cells (supporting hair cell function) for proper function. β-Catenin is an essential component of the Wnt/β-Catenin signaling pathway, which is required for normal development of different cell types. Here, we show that β-Catenin is required for differentiation of the supporting cell subtype inner pillar cells. Loss of transcriptional activity of β-Catenin caused loss of inner pillar cells and gain of outer pillar cell-like cells. In addition, we show that β-Catenin works in a cell autonomous manner within pillar cells to establish inner pillar cell identity. These findings advance our understanding of the mechanisms involved in cell identity establishment during cochlear development and thereby will guide regenerative efforts to repair damaged cells within the organ of Corti. [ABSTRACT FROM AUTHOR]

The mouse organ of Corti, housed inside the cochlea, contains hair cells and supporting cells that transduce sound into electrical signals. These cells develop in two main steps: progenitor specification followed by differentiation. Fibroblast Growth Factor (FGF) signaling is important in this developmental pathway, as deletion of FGF receptor 1 (Fgfr1) or its ligand, Fgf20, leads to the loss of hair cells and supporting cells from the organ of Corti. However, whether FGF20-FGFR1 signaling is required during specification or differentiation, and how it interacts with the transcription factor Sox2, also important for hair cell and supporting cell development, has been a topic of debate. Here, we show that while FGF20-FGFR1 signaling functions during progenitor differentiation, FGFR1 has an FGF20-independent, Sox2-dependent role in specification. We also show that a combination of reduction in Sox2 expression and Fgf20 deletion recapitulates the Fgfr1-deletion phenotype. Furthermore, we uncovered a strong genetic interaction between Sox2 and Fgf20, especially in regulating the development of hair cells and supporting cells towards the basal end and the outer compartment of the cochlea. To explain this genetic interaction and its effects on the basal end of the cochlea, we provide evidence that decreased Sox2 expression delays specification, which begins at the apex of the cochlea and progresses towards the base, while Fgf20-deletion results in premature onset of differentiation, which begins near the base of the cochlea and progresses towards the apex. Thereby, Sox2 and Fgf20 interact to ensure that specification occurs before differentiation towards the cochlear base. These findings reveal an intricate developmental program regulating organ of Corti development along the basal-apical axis of the cochlea. [ABSTRACT FROM AUTHOR]

Mammalian cochlear development encompasses a series of morphological and molecular events that results in the formation of a highly intricate structure responsible for hearing. One remarkable event occurs during development is the cochlear lengthening that starts with cochlear outgrowth around E11 and continues throughout development. Different mechanisms contribute to this process including cochlear progenitor proliferation and convergent extension. We previously identified that FGF9 and FGF20 promote cochlear lengthening by regulating auditory sensory epithelial proliferation through FGFR1 and FGFR2 in the periotic mesenchyme. Here, we provide evidence that ETS-domain transcription factors ETV4 and ETV5 are downstream of mesenchymal FGF signaling to control cochlear lengthening. Next generation RNA sequencing identified that Etv1 , Etv4 and Etv5 mRNAs are decreased in the Fgf9 and Fgf20 double mutant periotic mesenchyme. Deleting both Etv4 and Etv5 in periotic mesenchyme resulted in shortening of cochlear length but maintaining normal patterning of organ of Corti and density of hair cells and supporting cells. This recapitulates phenotype of mesenchymal-specific Fgfr1 and Fgfr2 deleted inner ear. Furthermore, analysis of Etv1/4/5 triple conditional mutants revealed that ETV1 does not contribute in this process. Our study reveals that ETV4 and ETV5 function downstream of mesenchymal FGF signaling to promote cochlear lengthening. • RNA sequencing reveals genes differentially expressed in Fgf9/20 mutant inner ears. • FGF9/20 regulates expression ETV1/4/5. • Loss of Etv4/5 in peri-otic mesenchyme shortens cochlear length. • Etv1 does not contribute cochlear length maintenance. [ABSTRACT FROM AUTHOR]