A MSTN^Del273C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia via MEK-ERK-FOSL1 axis

Joint Public Review:

Chen and collaborators first analysed in sheep embryonic gene editing using CRISPR-Cas9 technology to invalidate the two alleles of Mstn and Fgf5 genes by using different ratios of Cas9 mRNA and sgRNA. They showed that a ratio of 1:10 had highest efficiency and they successfully generated two sheep with biallelic mutations of both genes. Materials and Methods on the generation of gened edited sheep is entirely missing. The data on these gene edited sheep have been already published twice by the authors in different contexts. Other groups reported on gene editing of Mstn or Fgf5 in sheep embryos and the resulting phenotypes.

Although the findings are interesting, they do not provide sufficiently new scientific information or advancements in producing genetically modified livestock with improved production characteristics. While the MSTNDel273 sheep exhibited an increased number of muscle fibers, the data provided did not demonstrate a significant improvement in meat productions, quality or quantity in the MSTNDel273 sheep vs WT.

The authors indicate that sgRNA design changes in addition to changing the molar ratio of Cas9MRNA:sgRNA improved the ability to generate biallelic homozygous mutant sheep; however, the data provided to not demonstrate any significant difference. Given the small number of sheep that were actually produced and evaluated,it is extremely difficult to demonstrate anything that was analyzed to be significantly (statistically) different between MSTNDel273 sheep and WT, yet the authors seem to ignore this in much of their discussion. There is no explanation as to why the authors started with sheep that were FGF5 knockouts. The reviewer assumes that this was simply a line of sheep available from previous studies and the goal was to produce sheep with both improved hair/wool characteristics in addition to improved muscle development. However, the use of FGF5 knockout sheep complicates the ability to accurately decipher the unique aspects associated with targeting only myostatin for knock-out. At minimum, this is a variable that has to be considered in the statistical analysis. No information is provided on the methods used to produce the MSTNDel273 sheep, which is fundamentally important. It is assumed they were produced by injecting one-cell zygotes then transferring these into surrogate females. The methods employed might have a profound effect on the outcome.

Authors genotyped one sheep with a biallelic three base pair deletion in Mstn exon 3 and a compound heterozygote mutation in Fgf5 with a 5 nucleotides deletion on one allele and 37 nucleotides deletion on the other allele, partially spanning over the same region. This sheep developed a double muscle phenotype, which was documented using photography and CT scan. The hair phenotype was not further addressed, but authors referred to a previous publication.

Authors performed morphometric studies on two distinct muscles, longissimus dorsi and gluteus medius, and found a profound fiber hypotrophy in the Mstn-/-;Fgf5-/- double mutants, with a shift from larger fiber diameter to smaller fiber sizes. Morphometric studies showed only a low percentage of fibers in wt and mutant sheep had fiber cross sectional areas larger than 800 µm2, whereas about 30% in wt and about 60% in the mutant had CSA of <400 µm2. The report of one case, without reproducing the phenotype in other sheep, is scientifically insufficient. The fiber sizes in wt sheep remains far below previously published reports in sheep (about 3-5 times smaller) and as compared to other species, which suggests a methodological error in morphometric methods.

The authors also investigated the influence of Fgf5 mutation on muscle development. They determined fiber cross sectional area in heterozygous Fgf5 mutant (number of investigated animals not given) and conclude that Mstn mutation but not Fgf5 mutation caused the double muscle phenotype. Results are insufficient to support this conclusion. Firstly, authors investigated heterozygous FGF5 sheep and not homozygous mutants. Secondly, FGF5 has previously been shown to stimulate expansion of connective tissue fibroblasts and to inhibit skeletal muscle development during limb embryonic development (Clase et al. 2000). Of note, Mstn is also expressed during embryonic development. A combined knockout could therefore entail synergistic effects and cause muscle hyperplasia that is not found in individual knockout, a hypothesis that was not addressed by the authors.

The authors generated and studied an F1 generation of mutant sheep with heterozyogous mutation in Mstn and Fgf5. In Mstn+/-;Fgf5+/-, gluteus medius muscle was found to be larger compared to wt sheep, whereas other muscles were smaller, and overall meat quantity did not change. Morphometric studies revealed a similar muscle fiber hypotrophy and muscle hyperplasia as in the Mstn-/-;Fgf5-/- gluteus muscle.

In the next part of results, authors investigated the presence of myostatin protein in homozygous Mstn muscle using immunohistochemistry and found no differences compared to wt, however, positive and negative controls are missing. The also determined Mstn transcription and protein quantity using WB in heterozygous Mstn muscle and found no difference. The authors did not provide data to explain of why the herein generated Mstn mutation causes muscle fiber hypotrophy, whereas most work on myostatin abrogation demonstrated fiber hypertrophy.

Authors then isolated myoblasts from hind limbs of 3-month-old sheep fetuses and cultured in presence of 20% fetal bovine serum before switching to differentiation medium containing 2% horse serum. The cultures showed increased proliferation of Mstn+/-;Fgf5+/- myoblasts as well as downregulation of genes associated with muscle differentiation as well as reduced fusion index. No experiments were performed to assure whether the myostatin and FGF5 pathways were inhibited. No control experiments using supplementation with recombinant proteins and using growth factor depleted culture supplements were performed. As FGF5 and myostatin are secreted factors, evidence is missing whether this led to conditioning of the culture medium. Of note, previous work in mice demonstrated that the double muscle phenotype developed independent of satellite cells activity (Amthor et al. 2009).

Authors then performed RNA seq from Mstn+/-;Fgf5+/- muscle and found a number of differentially expressed genes, but none has been previously reported being involved in the myostatin signaling pathway, so the authors chose to only focus on FOSL1 and associated genes. Authors then demonstrated that Pdpn and Ankrd2 were upregulated during myogenic differentiation, whereas FOPSL1 was downregulated. Moreover, Fosl1 transcription was upregulated in myoblasts and myotubes from Mstn+/-;Fgf5+/- muscle. Authors showed an interaction between Fosl1 and Myod1. Moreover, authors demonstrated that Polsl1 directly binds to the Myod1 promoter. Authors also found decreased p38 MARPK protein levels in proliferating myoblasts from Mstn+/-;Fgf5+/- muscle and increased p38 MARPK in differentiating myotubes.

Furthermore, gain-of-function by overexpressing FOSL1 promoted cell proliferation and inhibited differentiation, and tert-butylhydroquinone, an indirect activator of FOSL1 also inhibited myogenic differentiation. The findings do not support the idea that FOSL1 is not involved, but neither do they strongly support the involvement of FOSL1. The observations made by the authors could be co-incidental and not causative in nature.

The manuscript by Chen et al. demonstrated successful gene editing in sheep embryos to obtain biallelic mutation of Mstn and FGF5. The resulting double muscle phenotype resulted from fiber hypotrophy and hyperplasia, which contradicts findings in the literature. Chen et al. generated F1 heterozygous offsprings, in which Mstn transcription and translation did not change. Myoblasts from these animals showed increased proliferation and decreased differentiation, which authors interpreted as the underlying cellular mechanism of the double muscle phenotype. However, no work on muscle development in these animals is presented. Important in vitro control experiments are missing. Chen and collaborators found Fosl1 as a differentially expressed gene in Mstn+/-;Fgf5+/- muscle. Fosl1 drives myoblast proliferation and has direct regulatory effect on the Myod1 promoter. The cellular and molecular mechanism of Fosl1 during myogenesis is novel and solid evidence. However, data remain inadequate to conclude whether Fosl1 indeed acts downstream of myostatin.

As the significant findings are minimal, the amount of text provided, figures and tables are disproportionally excessive. A large number of different molecular techniques are employed to try and decipher the mechanism(s) that result in the observed phenotype = double muscling. The authors focus on the MEK-ERK-FOSL1 pathway an suggest this the key pathway/mechanism resulting in the phenotype observed in MSTNDel273sheep. However, they provide very little solid evidence to support this notion.

The manuscript is very long, complicated and difficult to read, given the minimum amount of significant information that is provided. Further, it misses information in material methods, on the generation of animals, on histological techniques and morphometric studies. There is no information provided on the sex of the animals produced and then analyzed. There are also a number of editorial mistakes e.g. the authors refer to tables S1-S4 in the materials and methods and results section, but and there is no table S1-S4 provided.