基于Eph/ephrin通路研究Pou3f4对耳蜗蜗轴和骨螺旋板发育的调控机制

POU3F4 is the first gene found to cause X-linked nonsyndromic deafness. However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained unknown in a large part. Recent studies have indicated Pou3f4 may indirectly mediated fasciculation of SGNs with an Eph/ephrin pathway. The HRCT of the temporal bone in patients with POU3F4 gene mutations in our previous study suggests the absence of the cochlear modiolus and bony spiral lamina. Therefore, we propose a scientific hypothesis that SGNs can not be a bundle is a passive process, a secondary effect of abnormal development of cochlear modiolus and bony spiral lamina. Pou3f4 directly mediated the development of the cochlear modiolus and bony spiral lamina through the Epha4/ephrin pathway. In this study, we intend to verify this hypothesis by using Pou3f4 and Epha4 knockout mice as models and establishing an extracorporeal system through in vitro culture of mesenchymal cells. We intend to define the origin of developmental defects in the cochlear modiolus and bony spiral lamina, and the regulation of Pou3f4/EphA4 on mesenchymal cell condensation and cartilage differentiation in the cochlea, and the ephrin ligand involved in this process. These studies can expand the understanding of the regulation mechanism of POU3F4 transcription factor, and can also find a new mechanism for the regulation of the condensation and differentiation of mesenchymal cells in the development of inner ear bony labyrinth, therefore has important theoretical significance.

POU3F4是首个发现的导致X-连锁非综合征型耳聋的基因，但其作为转录因子调控的靶基因和发育过程在很大程度上仍然未知。近期有研究指出Pou3f4可能通过Eph/ephrin通路间接介导了SGNs成束。我们前期研究中收集到的POU3F4基因突变致聋患者影像学均提示蜗轴和骨螺旋板缺如。因此我们提出科学假说SGNs不能成束是一个被动的过程，是蜗轴和骨螺旋板发育异常的继发效应，Pou3f4通过Epha4/ephrin直接介导了蜗轴和骨螺旋板的发育。在本研究中，我们拟以Pou3f4和Epha4敲除小鼠以及体外培养的耳间充质细胞为模型，对这一假说进行验证。明确蜗轴和骨螺旋板发育缺陷的起源，Pou3f4/EphA4对耳间充质细胞聚集和软骨分化的调控，以及参与的ephrin。这些研究可以拓展对POU3F4转录因子调控机制的认识，还可以发现内耳骨迷路发育中间充质细胞聚集分化调控的新机制，具有重要的理论意义。

POU3F4是首个发现的导致X-连锁非综合征型耳聋的基因，但其作为转录因子调控的靶基因和发育过程在很大程度上仍然未知。已有研究指出Pou3f4通过Eph/ephrin通路介导了SGNs成束。我们前期研究中收集到的POU3F4基因突变致聋患者影像学均提示蜗轴和骨螺旋板缺如。因此提出科学假说SGNs不能成束是一个被动的过程，是蜗轴和骨螺旋板发育异常的继发效应，Pou3f4通过Epha4/ephrin直接介导了蜗轴和骨螺旋板的发育。在本研究中，我们成功构建Pou3f4y/-和Epha4-/-两种敲除小鼠模型。通过Pou3f4y/-敲除小鼠耳蜗Micro-CT扫描成像以及不同发育阶段的耳蜗切片染色和免疫荧光，发现Pou3f4y/-敲除小鼠耳蜗存在蜗轴及骨螺旋板发育缺陷，Pou3f4定位于形成蜗轴及骨螺旋板的耳间充质中，耳间充质与神经纤维空间互补定位，并为螺旋神经元神经纤维投射至耳蜗上皮提供物理支撑。其次，通过野生型小鼠不同发育阶段耳蜗石蜡切片染色，发现相同起源（间充质细胞）构成同一组织结构（耳蜗）的蜗壳和蜗轴最终是以不同方式成骨。Epha4-/-小鼠耳蜗石蜡切片染色结果表明，Epha4基因敲除小鼠耳蜗形态结构正常，说明Epha4基因不是Pou3f4介导蜗轴和骨螺旋板发育的直接靶DNA。通过Pou3f4y/-敲除小鼠和野生型小鼠耳蜗转录组测序-蛋白质谱关联分析获得的多个显著差异表达基因，GO富集分析发现骨矿化调节、骨发育、软骨发育等相关生物过程被显著富集。本项目研究成果明确了Pou3f4在耳蜗骨性结构发育过程中发挥着重要的转录调控作用，也为我们后续找到新的Pou3f4靶基因奠定了坚实的基础。