Wnt和Fgf信号通路在斑马鱼神经丘毛细胞再生中的相互作用及机制研究

The treatment of sensorineural hearing loss is still difficult and the promotion of damaged inner ear hair cell (HC) regeneration is an important solution. Supporting cell (SC) proliferation and hair cell transdifferentiation are the basic biological behaviors in HC regeneration. Thus, regulation of cell proliferation and differentiation becomes the key to promote regeneration after HC damage. Several studies have reported that Wnt signaling pathway can effectively induce the proliferation of SCs, while Fgf signaling pathway plays important role in HC differentiation. Our previous study observed that Wnt and Fgf signaling pathways were both involved in the process of HC regeneration. However, little is known about the role and mechanisms of Fgf signaling pathway in zebrafish HC regeneration, and the crosstalk between Wnt and Fgf signaling pathway during the process of HC regeneration remains undiscovered. These are all key issues for understanding the mechanisms of HC regeneration. In this study, we use zebrafish lateral line neuromast as experimental model to study the specific role of Wnt and Fgf signaling pathway in HC regeneration, and to further verify their interactions through epistasis experiments. At the same time, the molecular mechanisms and target point of their crosstalk will be explored by in situ hybridization and RNA-Seq assays, which finally provide new ideas and theoretical basis for HC regeneration of mammalian inner ear.

感音神经性聋的治疗目前仍是难题，促进受损的内耳毛细胞再生是重要的解决途径。支持细胞增殖和毛细胞转分化是毛细胞再生中的基本生物学行为，对细胞增殖和分化的调控成为促进毛细胞损伤后再生的关键。有研究报道Wnt信号通路能有效促进支持细胞的增殖，Fgf信号通路与毛细胞分化相关。我们前期研究观察到Wnt和Fgf信号通路均参与毛细胞再生进程，然而Fgf信号通路在斑马鱼毛细胞再生中的作用和分子机制目前并不清楚，Wnt和Fgf信号通路在毛细胞再生中是否有相互作用目前也未有报道。这些均是了解毛细胞再生机制的关键问题。在本课题研究中，我们将以斑马鱼侧线器神经丘毛细胞为研究对象，明确Wnt和Fgf信号通路在毛细胞再生中的具体作用和机制，进一步通过异位显性试验研究两者之间的相互作用，同时，通过原位杂交和RNA-Seq试验探索其相互作用的分子机制和靶点，并最终为哺乳动物内耳毛细胞再生提供新的思路和理论依据。

感音神经性聋的治疗目前仍是难题，通过促进细胞增殖和转分化维持一定数量的内耳毛细胞是保持听力的关键。Wnt和FGF信号通路在进化过程中高度保守，在多种器官比如听觉系统的发育中起着重要的调节作用，然而它们相互作用的分子机制尚不明确。在本研究中，我们使用斑马鱼侧线系统来研究Wnt和FGF通路在调节前体细胞增殖和再生细胞中的相互作用。我们发现激活Wnt信号通路可促进细胞增殖，同时增加发育及再生阶段神经丘毛细胞的数量。我们进一步证明，FGF信号通路在Wnt信号通路调节细胞增殖进程中起着至关重要的作用，抑制FGF信号通路可消除Wnt激活介导的细胞增殖效应，而用bFGF激活FGF信号则能部分解救因Wnt活性缺失导致的增殖减少和毛细胞数量缺失。斑马鱼胚胎整体原位杂交分析表明，当用Wnt信号通路激活剂BIO治疗后，一些FGF通路基因，包括pea3和fgfr1在神经丘的表达显著增加。有趣的是，当使用SU5402抑制FGF信号时，FGF信号通路受体基因fgfr1在神经丘的表达水平明显降低，但Wnt信号通路靶基因，包括ctnnb1、ctnnb2和tcf7l2的表达水平显著升高，而bFGF治疗不会改变这些基因的表达水平。以上结果表明在增殖过程中，fgfr1可能是Wnt信号通路的抑制剂。我们的研究结果表明，Wnt和FGF信号通路在斑马鱼早期发育以及新霉素诱导的毛细胞再生过程中相互作用，维持斑马鱼神经丘毛细胞的增殖和再生。