Regulation of Elongation, Growth and Coiling of the Mammalian Cochlea

蜗管的生长、延伸和卷曲是蜗管发育的关键步骤。 我们实验室和其他实验室的先前结果表明，一种进化上保守的称为平面细胞极性（PCP）通路的通路在耳蜗生长中起作用。 然而，PCP通路中的分子都不能产生耳蜗生长所需的分子力。 最近，一个特定的肌球蛋白分子，非肌肉肌球蛋白II，已被牵连作为PCP途径的效应。 因此，我们试图确定肌球蛋白II是否在耳蜗生长中发挥作用。 三种不同的肌球蛋白重链基因，肌球蛋白IIA，IIB和IIC的定位，表明肌球蛋白IIB和IIC都在发育中的耳蜗导管中表达。 此外，药理学抑制肌球蛋白II在体外抑制耳蜗生长，表明肌球蛋白II在此事件中起着重要作用。 为了证实肌球蛋白II的作用，我们表达了一个显性负性版本的肌球蛋白IIB专门在发展中的内耳。 使用显性阴性，因为肌球蛋白IIB或IIC的单一缺失由于两个基因之间的功能补偿而没有表型。 显性阴性肌球蛋白IIB的表达导致耳蜗缩短，这是由于导管内细胞生长缺陷的结果。 正在进行的实验将确定肌球蛋白II的分子作用和耳蜗生长的基础。 为了开始确定肌球蛋白II在耳蜗发育中的特定作用，使用针对肌球蛋白IIA、IIB或IIC的抗体来定位耳蜗内每种分子的表达。 结果表明，IIB和IIC的显着本地化的发展中的柱细胞，一个独特的支持细胞类型，位于内外毛细胞之间。 为了确定柱细胞是否可以发挥作用，在耳蜗的伸长，这些细胞的形状和位置进行了研究，在正常和肌球蛋白II突变耳蜗。 在正常耳蜗中，柱状细胞经历从圆形到细长的形状变化，细胞的伸长发生在沿着耳蜗延伸轴。 此外，发育中的柱细胞排列成一排，直接邻近发育中的内毛细胞。 相反，在肌球蛋白II突变体中，柱细胞不能伸长或排成一行。 这些结果表明，发育中的柱细胞可能在驱动发育中的耳蜗内细胞的伸长和图案化方面发挥关键作用。 组织延伸的另一个方面是细胞间粘附的表达和调节。 基于抗体标记，我们观察到一个独特的模式的表达细胞粘附分子，Ecad，在发展中的耳蜗。 特别是，Ecad在耳朵的区域中特异性表达，该区域将发育为外毛细胞。 为了确定Ecad是否在外毛细胞的伸长或图案化中发挥作用，我们在内耳内产生了Ecad的靶向缺失。 对这些突变小鼠的细胞模式分析表明，外毛细胞的排列发生了惊人的变化。 在Ecad突变体的耳朵中，外毛细胞排列成圆形，与正常耳朵中的三排不同，它们位于离正常位置一定距离的地方。 这些结果表明，Ecad和细胞粘附在外毛细胞的排列中起着关键作用。

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.