Thyroid hormone mediates fibroblast growth factor receptor expression, which both alter cell surface mechanical properties of the mammalian cochlea

Cells in the mammalian hearing organ, the cochlea, develop a specialized patterning and cellular architecture necessary for hearing function. Cytoskeletal components, such as actin and microtubules, maintain the material and mechanical properties of these cells. One way to understand cell material properties is to measure the response of the cytoskeleton to mechanical stress. Mechanical stress to sensory hair cells and nonsensory supporting cells in the cochlear epithelium, the organ of Corti, can lead to hearing loss, and currently these cells cannot recover from cytoskeletal damage. Quantification of the mechanical properties of these cells as they develop is lacking. By understanding cytoskeletal development, it may be possible to repair damaged cells to restore hearing function. Using Atomic Force Microscopy, we measured cell surface mechanical properties in cochlear tissue culture during development. We calculated and compared the Young's modulus of sensory outer hair cells (OHCs) and non-sensory supporting pillar cells (PCs) at discrete time points between embryonic day 16 (E16) and postnatal day 5 (P5). We find the first distinctions between OHC and PC Young's modulus in the late embryonic period, after E16. By P5, PCs have developed a network of acetylated microtubules that occupy the majority of PC cytoplasm, which may in part explain the higher Young’s modulus and increased stiffness relative to OHCs, which are primarily composed of actin filaments at the lumenal surface. To understand the growth factors regulating cochlear cellular development after E16, we antagonized both the Fibroblast growth factor (Fgf) and thyroid hormone (TH) signaling pathways. Using an in vivo mouse model, we find that loss of Fgfr3 leads to a decrease in the Young's modulus of both OHCs and PCs, which suggest that the disruptions to tissue architecture may contribute to the hearing loss in these animals. To disrupt TH levels in the cochlea, timed-pregnant female mice were treated with methimazole to induce a hypothyroid state in utero. We find that hypothyroid cochleae have a delay in down regulation of Fgfr3 in OHCs and disruptions to OHC and PC morphology. Interestingly, OHCs and PCs are stiffer in hypothyroid relative to control conditions. By examining both the transcriptional profile and the Fgf-signaling cascade, we find that the aberrant cellular stiffening results in part from persistent activation of Fgf-receptors and the actin depolymerizing factor cofilin. Finally, we wanted to examine the morphology and mechanics of OHCs and PCs before they are fully differentiated. Using immunohistochemistry and confocal microscopy, we find that overlap of prosensory specific markers, including Islet1, Sox2, and Jagged1, may help to refine the population of prosensory cells that will give rise to the organ of Corti. Defining this region may permit quantification of prosensory cell surface mechanical properties in the un-differentiated sensory epithelium. Together, these data suggest that growth factors impact the developing OHC and PC cytoskeleton, which dictates cell surface mechanical properties during development. By understanding the material properties of these cell types, future research may consider manipulation of growth factor signaling pathways to repair the organ of Corti in order to restore hearing function

Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP