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

• Applicant: Professor Dr. Said Hashemolhosseini
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 345115526

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.

• DFG Programme: Research Grants