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

The three severity levels of myopathic mouse phenotypes (Table 1) were analyzed for DUX4-dependent histopathology. The initial analysis of the mouse models showed different, muscle-specific levels of transgene recombination in all three bi-transgenic severity models ( Figure 1) . Therefore, cryosections for histological analysis were generated from multiple muscles, including TA, gastrocnemius, soleus, quadriceps, and heart, for all three models. These muscle sections were then analyzed by hematoxylin and eosin (H&E) staining to assess fiber morphology, TUNEL assay to assess apoptosis, embryonic myosin heavy chain (eMyHC/Myh3) IF to assess muscle fiber regeneration, picrosirius red (SR) staining to assess fibrosis, and CD11b and Ly6G IF to assess immune cell infiltration. The previously described time-courses of treadmill exhaustion running were performed using female mice for the moderate model, with (Figure 10) . Importantly, as seen in the mild model, these models showed anatomical muscle-specific differences in histopathogenic features, correlating with muscle-specific levels of transgene recombination ( Figure 1B) , consistent with DUX4-FL expression leading to an atrophic phenotype [34] . For example, the soleus muscle, which shows the highest level of TMX-responsive transgene recombination, also appears to have the greatest degree of histopathology for each model, including mononuclear cell infiltration, disrupted muscle integrity ( Figure 8K , O, S and Figure 9O , S), and fibers with centralized nuclei (Figure 10 ). The quadriceps showed the lowest level of transgene recombination and similarly showed the least amount of histopathology for the analyzed skeletal muscles. The heart, which showed no transgene recombination or expression, did not show any pathology even in the severe model ( Figure S6) . Overall, the skeletal muscle histopathology in these models was progressive, appearing with peak DUX4-FL expression levels at MD14 in the moderate model while appearing earlier in the severe model (SD6), and peaking with DUX4-FL levels at SD9. This further supports that the extent of pathology in these mouse models correlates with the level of transgene recombination ( Figure 1B ) and DUX4-fl expression (Figure 2 and 3) .