A new mouse model for FSHD

To understand the effect of DUX4 in vivo, and to generate a model in which to test anti-DUX4 therapeutics, we have introduced a doxycycline (dox)-inducible transgene encoding DUX4 and 3′ genomic DNA into a euchromatic region of the mouse X chromosome. Although this mouse was intended to provide doxycycline-inducible phenotypes, through expression of DUX4, we found that the transgene alone, without dox, was lethal in males. Females were runted and presented a skin pathology in the absence of dox. This is due to low level leaky expression of the DUX4 gene, as transcript can be detected by PCR in most tissues with the most consistent detection in neural tissues including retina, and in testis. We could not detect the protein in the absence of dox. Although males are not usually born live, occasional males survive to term. These animals are much more severely affected than their female counterparts, and remarkably, display a retinal vascular pathology similar to that seen in FSHD. In addition, they show a defect in spermatogenesis. Muscles from these animals were composed of smaller fibers and were weaker, but proportionally so, and no dystrophy was present prior to death, invariably before 6 weeks of age. We performed ex vivo culture experiments with myoblasts and muscle fibroadipogenic progenitors (FAPs) isolated from the iDUX4 males, and found that high levels of dox caused cell death of myoblasts and was growth inhibitory to fibroblasts. Low non-toxic levels of dox impaired differentiation of myoblasts in vitro, but had no effect on adipogenic differentiation of muscle FAPs. To evaluate the effect of DUX4 expression during regenerative repair in vivo, we crossed the Pax7-ZsGreen satellite cell marker into this strain and performed transplants of satellite cells from live-born males into female NSG-mdx4Cv mice,and treated recipients with varying doses of dox. By using dystrophin staining to quantify engraftment, we observed a consistent and easily quantifiable dose-dependent reduction in regeneration by transplanted satellite cells. These data show that DUX4 expression, even at low levels, is inhibitory to regenerative myogenesis. We further propose that the quantification of donor-derived myofibers generated by transplanted iDUX4 satellite cells can serve as a useful in vivo test system for therapeutics directed against the DUX4 protein or transcript