Matthew W. Kelley - US grants

Auditory and vestibular function are dependent of the formation of a functional inner ear. While there are multiple components for both of these systems, this laboratory focuses on the development of the sensory epithelia, which contain mechanosensory hair cells and associated cells called supporting cells and on the innervation of those hair cells by neurons from the VIIIth (acousticovestibular) cranial nerve. All three of these cell types are derived from the otocyst, a placodal structure that forms adjacent to the hindbrain early in development. Identifying the factors that specify each of these cell types and then direct their assembly into functional units is a key goal of the Section on Developmental Neuroscience. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. The ability of mammals, including humans, to discriminate a broad spectrum of frequencies is dependent on an elongated auditory sensory structure, called the organ of Corti, which extends along the entire length of the spiral of the cochlea. One of the most striking aspects of this organ is a precise alignment of mechanosensory hair cells into 4 rows that extend along the spiral. Previous studies from our laboratory and others have suggested that the precursor cells that give rise to hair cells may become organized into these rows through a conserved developmental process referred to as convergent extension (CE). However, the role of CE was only inferred through analysis of fixed tissue. To examine the role of CE in cochlear extension directly, we combined mouse genetics, in vitro explants and confocal live-imaging to study the outgrowth of the cochlear duct over time. Analysis of those movies demonstrated, as expected, that cochlear cells actively migrate outwards through the generation of cellular protrusions directed in the direction of migration. However, contrary to expectations, limited CE was observed. Instead analysis of the data indicated that radial intercalation (RI), the directed movement of cells towards the basement membrane, provides a significant driving force for cochlear extension. These results provide the first visualization of the cellular movements that drive cochlear extension. Moreover, they demonstrate that existing dogma regarding the processes that mediate cochlear outgrowth is incorrect. One hypothesis that arose from this study was the idea that outer hair cells become aligned, in part, as a result of restrictions in their ability to move. In particular, our results suggested that constraining developing hair cells along the medial-lateral axis to a relatively narrow region of epithelium would force their migration along the orthogonal basal-apical axis which could play a role in alignment. To test this, we are using cell-type specific mouse mutants to selectively eliminate constraint along the medial-lateral axis. Our prediction is that this change will lead to defects in outgrowth and alignment. In addition, another remarkable aspect of the cochlea is the precise coiling that occurs during development. We have begun to examine how selective growth of cells in different regions of the cochlea could play a role in inducing coiling. The results of this study should have implications in understanding human disorders that lead to shortened or misshapen cochleae such as Mondini Disorders or cochlear hypoplasias. A significant goal of the Section on Developmental Neuroscience is the characterization of cell types within the mammalian cochlea. In the past, a particular problem has been that there are a fairly large number of different cells types in the cochlea, at least 9 that we can identify based on morphology, but comparatively small numbers of any specific cell type. For instance there are only approximately 1000 inner hair cells in one mouse cochlea. These two conditions have made it difficult to develop transcriptional profiles for known or unknown cell types. The recent development of methods for isolation and capture of mRNAs from single cells offers an exciting opportunity to more fully characterize tissues like the cochlea. Therefore, we collected approximately 10,000 cells per time point at 6 different time points spanning cochlear development. The resulting data set allowed us to determine the total number of unique cells within the cochlea, about 18. Future analyses will allow us to determine genes and gene pathways that play a role in specifying many of those cell types. As part of our initial analysis of the single cell data, we were able to identify two genes, Lrrn1 and Sall1, that were expressed in subsets of cochlear cells. We then obtained or generated mutants for each of these genes and are presently analyzing the inner ear phenotypes in these animals. Finally, we have happily shared our expertise in single cell isolation and analysis with other laboratories at the NIDCD and NIH. This has resulted in the publication of several manuscripts over the past year with members of the Section on Developmental Neuroscience as authors.

Outgrowth, extension and coiling are key steps in the development of the cochlear duct. Previous results from our laboratory and others have indicated that an evolutionarily conserved pathway referred to as the planar cell polarity (PCP) pathway plays a role in cochlear outgrowth. However, none of the molecules in the PCP pathway act to generate the molecular force necessary for cochlear outgrowth. Recently, a specific myosin molecule, non-muscle myosin II, has been implicated as an effector of the PCP pathway. Therefore, we sought to determine whether myosin II plays a role in cochlear outgrowth. Localization of the three different Myosin heavy chain genes, Myosin IIA, IIB and IIC, indicated that Myosin IIB and IIC are both expressed in the developing cochlear duct. Moreover, pharmacological inhibition of myosin II in vitro inhibited cochlear outgrowth, indicating that myosin II plays an important role in this event. To confirm the role of myosin II, we expressed a dominant negative version of Myosin IIB exclusively within the developing inner ear. A dominant negative was used because single deletions of either Myosin IIB or IIC had not phenotype as a result of functional compensation between the two genes. Expression of dominant negative Myosin IIB leads to shortened cochleae as a result of defects in the growth of cells within the duct. Ongoing experiments will determined the molecular role of myosin IIs action and the basis for that action in cochlear outgrowth.

During the last year we have undertaken a detailed study of the signaling pathway that regulates the formation of one specific type of supporting cell, the pillar cell. Pillar cells are only found in mammalian inner ears and the presence of these cells is required for normal auditory function. An examination of expression of members of the fibroblast growth factor signaling pathway indicated that one fgf ligand, Fgf8 is expressed in a limited pattern of cells within the organ of Corti and that one of the fgf receptors, Fgfr3 is expressed in an adjacent population of cells. Interestingly, the cells that express Fgfr3 include cells that will develop as pillar cells. Previous work from the laboratory has demonstrated that deletion of Fgfr3 leads to a specific loss of pillar cells while over activation of Fgfr3 leads to increased pillar cell formation. These results suggest that expression of Fgfr3 plays a role in regulating the temporal and spatial position of pillar cells. To begin to understand how expression of Fgfr3 is regulated we examined the effects of thyroid hormone on Fgfr3 expression. Thyroid hormone signaling is known to regulate some aspects of Fgfr expression in other systems and the inner ear phenotype in thyroid hormone receptor mutants closely resembles the phenotype in Fgfr3 mutants. Initial studies have demonstrated that induction of either hyper- or hypo-thyroid conditions in mouse embryos results in specific changes in Fgfr3 expression with increased thyroid hormone signaling resulting in a down regulation of Fgfr3. Moreover, analysis of the Fgfr3 promoter indicates the presence of several thyroid hormone receptor binding sites, suggesting potential direct regulation of Fgfr3 expression by thyroid hormone. Ongoing experiments will determine whether these binding sites are valid and can regulate Fgfr3 expression. During an analysis of the effects of deletion of Fgfr3 we observed that Fgfr3-mutant cochleae contain a greater number of hair cells suggesting that some of the cells that would have developed as pillar cells have undergone a fate change to become additional hair cells. A screen for genes with altered expression in Fgfr3 mutants indicated that bone morphogenetic protein 4 (Bmp4) is up-regulated. Since Bmp4 has been shown to influence cell fate, we wanted to determine whether the increase in hair cells might be a result of the increase in Bmp4 signaling. To examine this possibility, Bmp4 signaling within the cochlea was modulated in vitro. Results indicated that increased Bmp4 leads to an increase in hair cells while inhibition of Bmp4 leads to hair cell loss. Moreover, the increased hair cell number in Fgfr3 mutant cochlea can be inhibited if Bmp4 signaling is blocked. These results suggest that a balance between Fgf and Bmp signaling may play a role in regulating the number of pillar cells versus hair cells within the developing organ of Corti. To begin to examine the specific role of Bmp4 in the formation of hair cells versus supporting cells, we first determined the expression of a family of transcription factors, referred to as Smads, that are activated in response to Bmp4 signaling. We found that multiple Smads are expressed in the same region of the ear as Fgfr3, suggesting that these two signaling pathways are active within the same cells. Moreover, using an antibody against the phosphorylated (activated) form of Smads1/5/8, we were able to demonstrate that Smads are activated in the same region of the inner ear in which Fgfr3 is activated. This result suggests that the balance of activated Bmp versus activated Fgf signaling pathways plays a key role in regulating the choice between hair cell and supporting cell. In order to test this hypothesis directly, we used and endogenous inhibitor of Bmp signaling, Noggin, to antagonize the amount of Bmp signaling within the inner ear. Preliminary results indicate that inhibition of Bmp signaling results in a dose dependent elimination of hair cells. These results support the hypothesis that the Bmp and Fgf signaling pathways interact within individual cells to determine whether those cells will form as hair cells or supporting cells.

In the first year of this project we initiated three sub-projects designed to provide a baseline understanding of how this system develops as well as to begin to identify at least some of the factors that mediate innervation within the cochlea. First, we began a descriptive study of the formation and differenation of spiral ganglion neurons using several antibody markers. Preliminary results indicate neuronal processes inter the developing cochlea prior to hair cell formation, but that the formation of specific contacts is dependent on the presence of developing hair cells. In a second series of experiments we used an genetic approach to label a small subset of developing spiral gangion neurons with yellow fluorescent protein. This approach will allow us to visualize single neurites as the establish connections with hair cells. In the future we will combine this technique with time lapse imagery to observe how specific connections are established Finally, as a third approach we have examined the effects of a population of mesenchymal cells that spiral ganglion neurites must pass through prior to entering the cochlea. Analysis of mice with a mutation in Pou3f4, a known deafness gene expressed exclusively in mesenchyme, indicates defects in neuronal pathfinding, indicating that mesenchymal cells play a key role in cochlear development.

? DESCRIPTION (provided by applicant): The Auditory Development: From Cochlea to Cognition Conference will provide a forum for junior and senior scientists from many disciplines to share unpublished, innovative research about the mechanisms that regulate the maturation of hearing at multiple levels of analysis. The conference will also address the impact of developmental research on our understanding of hearing disorders that arise from defects in mechanisms of maturation. The specific objectives are to foster interactions across sub disciplines, promote a sense of community, and encourage the development of collaborative research among those working on different aspects of auditory development. The conference will also provide a supportive scientific setting that will encourage and excite students to enter the field of auditory developmental research. The invited speakers include prominent scholars in the field, while the contributed presentations will feature promising young scientists who are applying new approaches or ideas to auditory developmental research. A second mechanism to encourage interaction and debate will be three round table discussions that each address a pressing question in the field. All participants will be encouraged to present their research at highly interactive, extended poster sessions, with 12 of the contributed abstracts selected for shorter talks that will be interleaved with invited presentations. The Auditory Development: Cochlea to Cognition Conference will be held August 14-15, 2015 at the University of Washington in Seattle, an ideal academic setting for encouraging intense and invigorating discussions in a collegial atmosphere. The specific program described in this application has the overarching goals of representing the breadth of developmental research in the hearing sciences, while also advancing new thematic foci. Thus, the Conference will highlight research areas that focus on potential interactions between auditory pathway loci. The influence of cognitive factors such as attention during the course of auditory system maturation will be considered. Finally, the meeting will integrate clinically relevant research in the area of hearing loss, including the vulnerability of both cochlea and central nervous system to developmental injury, and restoration. Here, basic research findings will dovetail with the latest findings on cochlear prostheses, regeneration, gene therapy and central plasticity mechanisms. The Conference will provide participants with an ideal setting to explore how research approaches at very different levels relate to one another, thereby facilitating new ideas and collaborations.