Precise alternating cellular pattern in the inner ear by coordinated hopping intercalations and delaminations.

The mammalian hearing organ, the organ of Corti, is one of the most organized tissues in mammals. It contains a precisely positioned array of alternating sensory hair cells (HCs) and nonsensory supporting cells. How such precise alternating patterns emerge during embryonic development is not well understood. Here, we combine live imaging of mouse inner ear explants with hybrid mechano-regulatory models to identify the processes that underlie the formation of a single row of inner hair cells (IHCs). First, we identify a previously unobserved morphological transition, termed "hopping intercalation," that allows cells differentiating toward IHC fate to "hop" under the apical plane into their final position. Second, we show that out-of-row cells with low levels of the HC marker Atoh1 delaminate. Last, we show that differential adhesion between cell types contributes to straightening of the IHC row. Our results support a mechanism for precise patterning based on coordination between signaling and mechanical forces that is likely relevant for many developmental processes.

A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Mechanical forces shaping the development of the inner ear.

The inner ear is one of the most complex structures in the mammalian body. Embedded within it are the hearing and balance sensory organs that contain arrays of hair cells that serve as sensors of sound and acceleration. Within the sensory organs, these hair cells are prototypically arranged in regular mosaic patterns. The development of such complex, yet precise, patterns require the coordination of differentiation, growth, and morphogenesis, both at the tissue and cellular scales. In recent years, there is accumulating evidence that mechanical forces at the tissue, the cellular, and the subcellular scales coordinate the development and organization of this remarkable organ. Here, we review recent works that reveal how such mechanical forces shape the inner ear, control its size, and establish regular cellular patterns. The insights learned from studying how mechanical forces drive the inner ear development are relevant for many other developmental systems in which precise cellular patterns are essential for their function.

Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness.

Genetic variants account for approximately half the cases of congenital and early-onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4-/- mice have severe-to-profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9-PHP.B, a recently developed synthetic adeno-associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4-/- mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory-associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations.

Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.