Dlx3b-4b和Gata3是斑马鱼内耳毛细胞发育的主要调节因子

Mechanosensory hair cells (HCs) of inner ear, delicate, fragile and sensitive to damaging factors, are trigger of hearing or vestibular neural circuitry. Heritable or gained HC defects often lead to otic dysfunctions. Otic HC development is under control of FGF, Bmp, Dlx3b-4b and Gata3 however, the underlying molecular genetic mechanisms are largely unknown. Our preliminary study reveals that Fgf-dependent otic Dlx3b and Gata3, when individually knocked down their functions, results in significant HC loss. Double knock down of both transcription factors completely abolishes HCs, indicating that Dlx3b and Gata3 are center to otic HC development program. We plan to use photo-cleavable morpholino oligos, inducible transgenic lines, CRISPRi/CRISPR-on strategy, small chemical inhibitors, TALENS and CRISPR/Cas9 genome editing tools, RNA-seq and ChIP-seq techniques to unveil a genetic regulation network directing otic HC specification. The network is based on Fgf，Bmp，Dlx3b-4band Gata3, and our proposed study will facilitate our understanding of sensual hearing or vestibular disorders, reveal novel regulators in the genetic network, and provide new insights of HC fate determination and helpful hints for HC regeneration study.

内耳毛细胞是听觉、平衡感觉神经环路的开关，极易损伤、死亡。先天性、获得性毛细胞缺失均导致内耳功能障碍。内耳毛细胞发育受不同信号和转录因子如Fgf， Bmp， Dlx3b-4b和Gata3等调控，但其机理还不十分清楚。敲低Fgf依赖的内耳Dlx3b或Gata3功能导致毛细胞减少，而双敲低导致无毛细胞，说明Dlx3b和Gata3是内耳毛细胞发育调控的核心。本研究计划运用光切割缓释型morpholino技术，诱导型转基因鱼系，CRISPRi/CRISPR-on技术，小分子阻断化合物，TALENs 或CRISPR/Cas9基因组修饰技术、RNA-Seq和ChIP-Seq等手段全面阐明一个基于Fgf，Bmp，Dlx3b-4b和Gata3的斑马鱼内耳毛细胞发育调控网络。本研究有助于加深理解听力、平衡障碍成因，揭示内耳毛细胞命运决定机制和调控新基因，并为内耳毛细胞再生研究提供理论依据。

本研究运用多种基因干扰/编辑技术调节dlx3b表达，证明Dlx3b调控内耳毛细胞发育，初始毛细胞（tether cell）/耳石形成，后两者可能影响前庭功能-通过自制前庭眼动反射（VOR）仪检测。尽管dlx3b和dlx4b互为旁系同源基因，单独敲降/敲除dlx4b并不影响毛细胞发育和耳石形成；只有同时缺失dlx3b和dlx4b功能才导致dlx3b突变体内耳毛细胞进一步减少，耳石消失；而Dlx3b直接结合dlx4b的调控区域，抑制dlx4b表达。因此，Dlx4b只能一定程度上补偿Dlx3b在内耳发育中的作用。本研究还发现Gata3也参与斑马鱼内耳毛细胞发育但不影响耳石形成；Dlx3b和Gata3功能同时缺失则导致内耳毛细胞几近消失。发育阶段性敲降dlx3b或gata3的原位表达实验揭示Dlx3b较早（10-14hpf）参与而Gata3较晚（13-16hpf）介入调控内耳毛细胞发育。尽管阻断Fgf信号通路造成dlx3b和gata3在耳基板区域的表达显著降低，而Dlx3b和Gata3正向调控前神经基因atoh1表达，但稍后阻断Fgf（16-18hpf）几乎不影响内耳毛细胞发育，这与 Wnt信号自15hpf始介入atoh1表达调节契合。总之，斑马鱼内耳atoh1表达是由Fgf, Dlx3b/Dlx4b, Gata3 和Wnt/β-catenin有序地开启与维持，确保内耳毛细胞形成。本研究首次全面阐述了斑马鱼内耳毛细胞和耳石发育的分子遗传学机制及其与前庭功能的关联。.此外，本项目还发现在胚胎原肠至神经发育早期，Foxi1，Wnt和Fgf的有序介入决定了神经边缘区细胞特化出内耳细胞；成功地鉴定出micall2b，kcna3等基因为ADHD致病基因。