Regulation of Elongation, Growth and Coiling of the Mammalian Cochlea

耳蜗管的生长、延伸和盘绕是耳蜗管发育的关键步骤。我们实验室和其他人先前的结果表明，一种进化上保守的途径被称为平面细胞极性（PCP）途径在耳蜗生长中起作用。然而，PCP通路中的任何分子都不能产生耳蜗生长所需的分子力。最近，一种特殊的肌球蛋白分子，非肌肉肌球蛋白II，被认为是PCP途径的效应物。因此，我们试图确定肌球蛋白II是否在耳蜗生长中起作用。3种不同的肌球蛋白重链基因Myosin IIA、IIB和IIC的定位表明，Myosin IIB和IIC都在发育中的耳蜗中表达。此外，体外肌球蛋白II的药理抑制抑制了耳蜗的生长，表明肌球蛋白II在这一事件中发挥了重要作用。为了证实myosin II的作用，我们在发育中的内耳中表达了myosin IIB的显性阴性版本。使用显性阴性是因为肌球蛋白IIB或IIC的单个缺失由于两个基因之间的功能补偿而没有表型。显性负Myosin IIB的表达导致耳蜗导管内细胞生长缺陷导致耳蜗缩短。正在进行的实验将确定肌球蛋白ii的分子作用及其在耳蜗生长中的作用基础。

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. An additional aspect of cochlear outgrowth is the surprising link between growth of the duct and the presence and normal function of cilia on cells within the duct. In humans, defects in cilia growth or function result in a conserved cluster of developmental defects that include, among other things, visual and auditory impairment. In collaboration with the laboratory of Anand Swaroop at NEI we have examined interactions between genes that are known to regulate cilia development and genes that must be transited along the cilia for normal cellular function. The results of these experiments suggest that routing of specific proteins along the cilia is a crucial step in the formation and function of a number of cell types including both inner ear hair cells and retinal photoreceptors. A more complete understanding of how these interacting proteins regulate cellular functions should provide a greater understanding of the underlying pathogenesis in individuals with mutations in these genes.