The goal of the Section on Developmental Neuroscience is to identify the cellular, molecular and genetic factors that play a role in the development of the sensory epithelium of the mammalian cochlea, the organ of Corti. The organ of Corti is comprised of a highly rigorous pattern of specialized cell types that has been shown to be required for normal hearing. During the last year, members of the laboratory concentrated on several different issues related to the development of the organ of Corti. ? Previous results from our laboratory and other laboratories had identified the basic helix-loop-helix transcription factor (bHLH) Math1 as a key regulator of the development of mechanosensory hair cells. However the factors that regulate expression of Math1 are unknown. To begin to understand how Math1 expression is regulated, we examined the expression and role of a group of molecules called Ids (Inhibitors or differentiation). Ids are related to Math1 and other bHLH genes but cannot act as transcription factors because they lack a DNA binding domain. Instead, Ids serve as antagonists of bHLH molecules through a competitive interaction for a required common binding partner. Therefore, we wondered whether Ids might act to regulate the timing of Math1 activity. The Id family contains four separate molecules generated from four unique genes. Initial PCR screening indicated that three of these, Id1, Id2 and Id3 are expressed in the developing cochlea. Moreover, expression of all three Ids was down-regulated in cells that would develop as hair cells at about the same time that Math1 became active in those cells. To confirm that Ids were acting to antagonize Math1, gene transfer was used to prolong the expression of Id molecules beyond the normal period of down-regulation. Analysis of the fates of cells that maintained expression of Ids indicated that these cells were inhibited from developing as hair cells. These results identify Ids as important regulators of Math1 activity and, therefore, of hair cell formation in the cochlea.? In a separate series of experiments we began to examine the mechanisms through which Vangl2 regulates the generation of uniform orientation of stereociliary bundles within the cochlea. All mechanosensory hair cells have a specialized bundle of actin-based cilia, referred to as stereocilia, located on the luminal surface of the cell. Deflection of this bundle in a particular direction is perceived by that cell as sound. However, each hair cell is only sensitive to deflections of the bundle in a single direction. Therefore, the establishment of appropriate directional orientation of each bundle is crucially important for function. Previous results from our lab had demonstrated that there are significant defects in stereociliary bundle orientation in animals carrying a mutation in Vangl2. However the bases for these defects were unknown. To begin to determine the role of Vangl2, we generated an antibody against the protein. This antibody was then used to localize Vangl2 protein within developing hair cells. Our results indicated that Vangl2 becomes asymmetrically localized to the side of each hair cell opposite to the final location of the stereociliary bundle. In addition, we demonstrated that asymmetric localization of Vangl2 is lost in mice with mutations in molecules that have been shown to interact with Vangl2. We also observed a direct correlation between the degree of loss of asymmetry and the degree of stereociliary bundle mis-orientation. Finally, we were able to demonstrate protein-protein interactions between Vangl2 and two potential interacting molecules, Scrb1 and Fz3. These results begin to identify the molecule components that are required for uniform stereociliary bundle orientation and provide us with insights into the molecular pathways that are required for the uniform orientation of cells within an epithelium, a phenomenon which is required for the development of multiple aspects of the vertebrate body plan.
Showing the most recent 10 out of 38 publications
