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.? ? To begin to determine the specific role of Myosin II in cochlear development, antibodies against Myosin IIA, IIB, or IIC were used to localize expression of each molecule within the cochlea. Results indicated a striking localization of both IIB and IIC to the developing pillar cells, a unique supporting cell type that is located between the inner and outer hair cells. To determine whether pillar cells could play a role in cochlear elongation, the shape and position of these cells was studied in normal and Myosin II mutant cochleae. In normal cochlear, pillar cells undergo a change in shape from rounded to elongated with the elongation of the cell occurring along the axis of cochlear extension. In addition, developing pillar cells become aligned in a single row located directly adjacent to the developing inner hair cells. In contrast, in Myosin II mutants, pillar cells fail to elongate or to become aligned in a single row. These result suggest that developing pillar cells could play a key role in driving the elongation and patterning of cells within the developing cochlea.? ? Another aspect of tissue extension is the expression and regulation of adhesion between cells. Based on antibody labeling, we observed a unique pattern of expression for the cellular adhesion moledule, Ecad, within the developing cochlea. In particular, Ecad is specifically expressed in the region of the ear that will develop as the outer hair cells. To determine whether Ecad could play a role in the elongation or patterning of outer hair cells, we generated a targeted deletion of Ecad within the inner ear. Analysis of the cellular patterning in these mutant mice indicated a striking change in the arrangement of outer hair cells. Instead of being aligned into three rows, as would be present in a normal ear, in Ecad mutant ears, outer hair cells are arranged in circular rosettes located at a distance from their normal location. These results suggest that Ecad and cellular adhesion play key roles in the alignment of outer hair cells.
| Yamamoto, Norio; Okano, Takayuki; Ma, Xuefei et al. (2009) Myosin II regulates extension, growth and patterning in the mammalian cochlear duct. Development 136:1977-86 |
