The neuromuscular role of canonical Wnt and/or YAP/Taz/Tead signaling

Wnt通过典型Wnt/b-连环蛋白依赖和非典型信号通路调节发育和分化等过程。另一个参与控制器官大小、组织再生和干细胞自我更新的重要途径是Hippo途径，其信号成员为YAP/Taz和Tead。最近，我们利用Axin2-lacZ报告小鼠阐明了典型Wnt活性在成人肌纤维中的作用。在这些小鼠中，活跃的典型Wnt信号通过受Axin2启动子控制的lacZ表达来反映，Axin2启动子本身是一个靶基因，也是典型Wnt信号的负调节因子。我们在培养的肌管中检测到活跃的典型Wnt信号(1),(2)在纤维直径较小的IIa和IIx型肌纤维中，(3)在神经肌肉连接处（NMJ），以及(4)在损伤后骨骼肌再生过程中。有趣的是，我们首次发现YAP/Taz/ tead1介导的信号传导在成人肌纤维中伴随着典型的Wnt信号传导。在培养的肌肉细胞中，(1)缺乏Axin1干扰增殖，(2)缺乏Axin2减慢向肌管的分化，(3)敲除b-catenin或Tead1后，肌生成增加。此外，典型Wnt3a在培养的肌肉细胞中诱导TOPflash和Tead1报告基因。现在，我们的目标是了解典型Wnt和YAP/Taz/Tead信号在神经肌肉环境中的完整作用，例如肌肉细胞，神经末梢和终末雪旺细胞。我们将确定典型Wnt和YAP/Taz/Tead在所有细胞NMJ部位的表达谱，但进一步关注这些信号通路的生物学作用，主要是在静止的成人肌纤维中，在心脏毒素损伤后的再生中，以及在NMJ的形成和维持中。为此，我们将使用带有Axin1、Axin2、Axin1/2、Lrp5/6、YAP/Taz和Tead等固定等位基因的转基因小鼠。作为Cre小鼠，我们将使用Pax7-CreERT2去除肌肉卫星细胞中的弯曲等位基因，HSA（人骨肌动蛋白）-Cre去除成人肌纤维中的弯曲等位基因，HB9-Cre去除运动神经中的弯曲等位基因。我们将通过它们的行为、握力测量和肌肉切片的组织学/组织化学/免疫染色来分析所有突变小鼠的肌肉表型。我们将通过结构和功能方法研究突变小鼠的NMJs，如3D形态测量成像和神经肌肉传递的电生理记录。NMJs的进一步方面，如形成和维持，将通过原代培养的肌管进行研究。通过RNA-Seq分析典型Wnt和YAP/Taz/Tead信号的肌肉靶基因。最后，我们将研究为什么这两种信号通路在骨骼肌中以纤维类型依赖的方式起作用。该项目将有助于了解神经肌肉生物学中两种信号通路的个体和协同作用。

Wnts regulate processes such as development and differentiation by canonical Wnt/b-catenin dependent, and non-canonical signaling pathways. Another important pathway involved in the control of organ size, tissue regeneration and stem cell self-renewal is the Hippo pathway, with its signaling members YAP/Taz and Tead.Recently, we elucidated the role of canonical Wnt activity in adult muscle fibers using Axin2-lacZ reporter mouse. In these mice, active canonical Wnt signaling is reflected by lacZ expression under control of the Axin2 promoter, which itself is a target gene and negative regulator of canonical Wnt signaling. We detected active canonical Wnt signaling (1) in cultured myotubes, (2) in muscle fibers with small fiber diameter and belonging to fiber types IIa and IIx, (3) at neuromuscular junctions (NMJ), and (4) during regeneration of skeletal muscle after injury. Interestingly, we showed for the first time that YAP/Taz/Tead1-mediated signaling accompanied canonical Wnt signaling in adult muscle fibers. In cultured muscle cells, (1) absence of Axin1 interfered with proliferation, (2) absence of Axin2 slowed down differentiation into myotubes, and (3) after knockdown of either b-catenin or Tead1 myogenesis was increased. Moreover, canonical Wnt3a induced TOPflash and Tead1 reporters in cultured muscle cells.Now, we aim to understand the complete role of canonical Wnt and YAP/Taz/Tead signaling in the neuromuscular context, e.g. muscle cells, nerve endings and terminal Schwann cells. We will identify expression profiles of canonical Wnt and YAP/Taz/Tead in all cellular NMJ parts, but focus further on elucidation of the biological role of these signaling pathways mostly in resting adult muscle fibers, in regeneration after cardiotoxin injury, and regarding NMJ formation and maintenance. To this end, we will use genetically modified mice with floxed alleles for Axin1, Axin2, Axin1/2, Lrp5/6, YAP/Taz, and Tead. As Cre mice, we will deploy Pax7-CreERT2 to delete floxed alleles in muscle satellite cells, HSA (human skeletal actin)-Cre to ablate floxed alleles in adult muscle fibers, and HB9-Cre to remove floxed alleles in motor nerves. We will analyze muscle phenotypes of all mutant mice by their behavior, grip strength measurements, and histological / histochemical / immunostainings of muscle sections. We will investigate the NMJs of the mutant mice by structural and functional approaches, like 3D morphometrical imaging, and electrophysiological recordings of neuromuscular transmission. Further aspects of NMJs, like formation and maintenance, will be studied by primary cultured myotubes. Muscular target genes of canonical Wnt and YAP/Taz/Tead signaling will be analyzed by RNA-Seq. Finally, we will investigate why both signaling pathways act in a fiber type dependent manner in skeletal muscles. This project will help to understand individual and concerted roles of both signaling pathways regarding neuromuscular biology.