Generation and characterization of a novel gne Knockout Model in Zebrafish.

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knocked-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8-10 days post-fertilization (dpf), but a phenotype appeared less than 24 h before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets.

In vivo and in vitro genome editing to explore GNE functions.

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, no reliable animal model is available for the disease and cellular models from GNE myopathy patients' muscle cells (expressing the mutated protein) are less informative than expected. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

The Diversity of Muscles and Their Regenerative Potential across Animals.

Cells with contractile functions are present in almost all metazoans, and so are the related processes of muscle homeostasis and regeneration. Regeneration itself is a complex process unevenly spread across metazoans that ranges from full-body regeneration to partial reconstruction of damaged organs or body tissues, including muscles. The cellular and molecular mechanisms involved in regenerative processes can be homologous, co-opted, and/or evolved independently. By comparing the mechanisms of muscle homeostasis and regeneration throughout the diversity of animal body-plans and life cycles, it is possible to identify conserved and divergent cellular and molecular mechanisms underlying muscle plasticity. In this review we aim at providing an overview of muscle regeneration studies in metazoans, highlighting the major regenerative strategies and molecular pathways involved. By gathering these findings, we wish to advocate a comparative and evolutionary approach to prompt a wider use of "non-canonical" animal models for molecular and even pharmacological studies in the field of muscle regeneration.

Zebrafish models of sarcopenia.

Sarcopenia - the accelerated age-related loss of muscle mass and function - is an under-diagnosed condition, and is central to deteriorating mobility, disability and frailty in older age. There is a lack of treatment options for older adults at risk of sarcopenia. Although sarcopenia's pathogenesis is multifactorial, its major phenotypes - muscle mass and muscle strength - are highly heritable. Several genome-wide association studies of muscle-related traits were published recently, providing dozens of candidate genes, many with unknown function. Therefore, animal models are required not only to identify causal mechanisms, but also to clarify the underlying biology and translate this knowledge into new interventions. Over the past several decades, small teleost fishes had emerged as powerful systems for modeling the genetics of human diseases. Owing to their amenability to rapid genetic intervention and the large number of conserved genetic and physiological features, small teleosts - such as zebrafish, medaka and killifish - have become indispensable for skeletal muscle genomic studies. The goal of this Review is to summarize evidence supporting the utility of small fish models for accelerating our understanding of human skeletal muscle in health and disease. We do this by providing a basic foundation of the (zebra)fish skeletal muscle morphology and physiology, and evidence of muscle-related gene homology. We also outline challenges in interpreting zebrafish mutant phenotypes and in translating them to human disease. Finally, we conclude with recommendations on future directions to leverage the large body of tools developed in small fish for the needs of genomic exploration in sarcopenia.

Gne depletion during zebrafish development impairs skeletal muscle structure and function.

GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. The process by which GNE mutations lead to myopathy is not well understood. By in situ hybridization and gne promoter-driven fluorescent transgenic fish generation, we have characterized the spatiotemporal expression pattern of the zebrafish gne gene and have shown that it is highly conserved compared with the human ortholog. We also show the deposition of maternal gne mRNA and maternal GNE protein at the earliest embryonic stage, emphasizing the critical role of gne in embryonic development. Injection of morpholino (MO)-modified antisense oligonucleotides specifically designed to knockdown gne, into one-cell embryos lead to a variety of phenotypic severity. Characterization of the gne knockdown morphants showed a significantly reduced locomotor activity as well as distorted muscle integrity, including a reduction in the number of muscle myofibers, even in mild or intermediate phenotype morphants. These findings were further confirmed by electron microscopy studies, where large gaps between sarcolemmas were visualized, although normal sarcomeric structures were maintained. These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity.

The change in genetic diversity down the core-edge gradient in the eastern spadefoot toad (Pelobates syriacus).

Several hypotheses are available to predict change in genetic diversity when approaching peripheral populations. We used the eastern spadefoot toad in Israel as a model system to examine these hypotheses using population genetics analyses and network theory. Our results contradicted most of the predictions from the 'abundant centre' model, that edge populations should have lower density and lower genetic diversity than core populations. Furthermore, dispersal rate between core and peripheral populations is expected to be asymmetric, mostly directed outwards from the core population, but we did not detect such a trend. Our data did not support the hypothesis of no change or a non-linear change in genetic diversity towards the range edge. However, our results did fit the Fisher (The Genetical Theory of Natural Selection, Clarendon Press, Oxford, 1930) hypothesis, which predicts increase in genetic variability from core to edge of distribution. We attributed this finding to the much harsher climatic and abiotic conditions at the edge, which must be tolerated over generations by both tadpoles and post-metamorphic individuals in this region. Finally, our results have significant conservation implications for the survival of this species in Israel, where it is critically endangered. We identified two distinct communities, which are genetically linked through two specific rain pools in the Upper Galilee. Details on the spatial subdivision of this species are cardinal for future management and restoration of temporary wetlands in Israel.