Abstract
Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN−/− F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN−/− F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.
Highlights
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and is a well-known negative regulator of myogenesis in skeletal muscle development[1,2,3,4,5]
Because MSTN is known to activate the Smad2/3 pathway, which in turn leads to the down-regulation of myogenic related factors (MRFs), we investigated the expression of the Smad2/3-regulated MRFs in MSTN−/− F4 medaka from 1 to 8 wph. mRNA was extracted from whole wild type (WT) and MSTN−/− F4 medaka and subjected to real-time PCR to detect the expression of three major MRFs: Myf[5], Myogenin and MyoD22, 27
Other platforms have been used to generate MSTN-mutated fish, to our knowledge, this is the first investigation of the MSTN−/− phenotypes produced by the CRISPR/Cas[9] system in medaka, and the first to document the MSTN−/− fish for several generations
Summary
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and is a well-known negative regulator of myogenesis in skeletal muscle development[1,2,3,4,5]. Most fish species carry two or more copies of MSTN In teleosts, this has led to a diversification in MSTN functionality and/or mechanisms, and has given rise to various phenotypes in which changes in growth performance and skeletal muscle development can occur via both hyperplasia and/or hypertrophy[21,22,23,24,25]. This has led to a diversification in MSTN functionality and/or mechanisms, and has given rise to various phenotypes in which changes in growth performance and skeletal muscle development can occur via both hyperplasia and/or hypertrophy[21,22,23,24,25] This diversity means that there is no universal fish research model for MSTN. Since there is often a trade-off between growth and other biological traits, such as immunity[32] and reproduction[33], we evaluate virus susceptibility by challenging the MSTN−/− medaka with RGNNV (red spotted grouper nervous necrosis virus) and assess their reproductive performance by monitoring both the number of eggs produced and the number of eggs that were successfully fertilized
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