Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

Takako I. Jones; Guo-Liang Chew; Pamela Barraza-Flores; Spencer Schreier; Monique Ramirez; Ryan D. Wuebbles; Dean J. Burkin; Robert K. Bradley; Peter L. Jones

Modeling FSHD in transgenic mice has historically been very difficult despite the fact that FSHD is a gain-of-function disease seemingly amenable to transgenic DUX4 overexpression [68, 74, 76] . While the human DUX4 gene has a conserved developmental role with Dux family members found in other mammals, including the mouse, there is significant divergence at the DNA and protein sequence level as well as in the spectrum of species-specific target genes [23, 70, 104] . In addition, because DUX4-FL is highly cytotoxic for many somatic cells, leaky expression during development has been problematic, and surviving mice can be severely phenotypic and difficult to breed [75] . However, the FLExD mice we recently developed overcame many of these previous limitations [41] . Both male and female mice are fertile and easy to breed, male or female transgenic mice can be produced either as transgene heterozygotes or homozygotes, which can live more than 1.5 years, and, when mated with an inducible Cre line of mice, bi-transgenic mice allow for investigator-controlled expression of DUX4-fl mRNA and protein resulting in FSHD-like muscle pathology [41] . Here we report the characterization of a series of phenotypic FSHD-like mouse models varying in severity from mild to severe, generated using the FLExD conditional DUX4-fl transgenic mouse line crossed with ACTA1-MCM TMXinducible mice. We demonstrate that these bi-transgenic mouse models, with and without TMX induction, recapitulate many aspects of FSHD pathophysiology with variable severity, thus providing suitable models for therapeutic interventions targeting DUX4-fl mRNA, protein, and certain downstream pathways, with several key caveats. In particular, it is important for those working on these models to keep in mind the described anatomical muscle-specific and sexspecific differences in pathology and disease progression. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/471094 doi: bioRxiv preprint Cre activity in the absence of TMX in a fraction of nuclei. Thus, in the ACTA1-MCM;FLExD bi-transgenic mice, this results in recombination of the DUX4 transgene and low mosaic expression of DUX4-FL protein in skeletal muscles throughout their lifetime. Importantly, although this leaky recombination is specific to skeletal muscle, it is not uniform among skeletal muscles, with different anatomical muscles exhibiting different, but consistent, levels of recombination and thus DUX4-fl expression ( Figure 1B , e.g. soleus is low and quadriceps is high). Thus, the situation in this mild model is similar to that in pre-symptomatic or asymptomatic FSHD patients. These mice live with a chronic, low-level, mosaic expression of DUX4-FL in a fraction (<10%) of muscle fibers, yet phenotypically the mice appear healthy and behave normally, with no changes in overall fitness or lifespan.