Transcriptomic and morphological characterization of a first Zebrafish model of Limb Girdle Muscular Dystrophy D2

Limb Girdle Muscular Dystrophy D2 (LGMD D2) is a rare neuromuscular disorder caused by a heterozygous mutation in the termination codon of the TNPO3 gene. This gene encodes for Transportin-3 (TNPO3), an importin which normally mediates the translocation to the nucleus of SR proteins, a family of splicing factors and other proteins related to RNA metabolism. LGMD D2 is characterized by high variability in the onset and progression of the disorder, and the main clinical features are progressive muscle weakness and marked atrophy. Several mutations have been described as causative of LGMD D2, all resulting in a mutated protein that is 15-aminoacids longer in its C-terminal domain. However, the pathogenetic mechanism of this disorder remains unknown. This project aims at investigating the pathogenesis of LGMD D2 utilizing an in vivo approach based on the creation of a Zebrafish model of disease. We focused on the role of TNPO3 on muscle development to identify any potential molecular pathways linked to the disorder. Experimentally we proceeded with the microinjection of mRNAs encoding the wild type or mutated form of human TNPO3 into Zebrafish embryos, to follow their effects on the myogenic processes during development up to 48 hpf. The subsequent analysis showed abnormalities in the gene expression profiles of Myogenic Regulatory Factors (MRFs) and muscle-specific proteins, suggesting an unbalance in the normal myogenic process. Next, we analysed how the transcriptomic alterations were reflected at a morphological level by transmission electron microscopy studies. At ultrastructural level we observed a random organization of myofibrils limited to the embryos microinjected with the mutant form of human TNPO3. These results suggest that our approach could be effective in establishing a Zebrafish model of LGMD D2 and enabled us to gain an initial understanding of the role of TNPO3 in muscle development and the pathogenic mechanism of LGMD D2.