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 FLExD hemizygous mice showed no transgene recombination in the absence of Cre, and the bi-transgenic animals showed variable low levels (2-10%) of transgene recombination in skeletal muscles, but not in heart or liver, in the absence of TMX, due to the above-mentioned sporadic leaky nuclear Cre activity in ACTA1-MCM mice. When injected with TMX to induce Cre nuclear activity and stimulate transgene recombination in skeletal muscles, the bi-transgenic mice showed increased recombination (8-30%) in response to the low TMX dose and an even higher level of recombination (38-55%) in response to the high TMX dose. Surprisingly, in all three bi-transgenic models (no, low, and high TMX) there were muscle-specific differences in the transgene recombination rate; however, these differences were consistent between the TMX treated lines ( Figure 1B ). For example, quadriceps muscles showed the lowest recombination in both TMX-induced models, followed by the tibialis anterior (TA) and gastrocnemius muscles with intermediate levels, while the diaphragm and soleus showed the highest recombination levels. As expected when using the ACTA1-MCM driver line, the heart and liver showed no transgene recombination in any of the bi-transgenic animals. Since this assay measures transgene recombination of a single copy transgene per nucleus, the results showing less than 100% recombination represent mosaic recombination which should translate into the desired mosaic DUX4-fl transgene expression. We conclude that we have identified three conditions that reproducibly produce differing levels of mosaic transgene recombination, which we will refer to as mild, moderate, and severe (Table 1) , based on the subsequent characterizations described below. Importantly for future studies, we show that different skeletal muscles show different levels of recombination in response to these TMX treatments. To assess if the variable rates of transgene recombination in each model translated similarly to variable levels of protein expression at the single nucleus level, muscle tissues were analyzed by immunofluorescence (IF) using an anti-DUX4-FL antibody. Mosaic patterns of nuclear DUX4-FL expression were readily detected in cross sections of TA muscle from all three models ( Figure 2 ). The bi-transgenic ACTA1-MCM;FLExD mice showed few DUX4-FL positive nuclei in the absence of TMX ( Figure 2M and N), consistent with a very low level of transgene recombination ( Figure 1B) . However, since DUX4-FL is cytotoxic and it is not known how long DUX4-FL expressing cells may remain in the muscles, an IF time course study was performed for the TMX-injected mice. The low-dose mice, which appeared to be moderately affected phenotypically over time and will be referred to as the moderate FSHD-like model (Table 1 , discussed in detail below), were assayed over 28 DPI. DUX4-FL expression appeared by 3 DPI (moderate day 3, or MD3), peaking at MD14, and then was greatly reduced by MD28, likely due to the death of DUX4-positive cells (Figure 2A -J). Mice injected with the high-dose TMX regimen were so severely affected by 9 DPI that they had to be sacrificed and could not be assessed further. These mice, which will be referred to as the severe FSHD-like model, similarly showed DUX4-FL expression by three days after the first injection (severe day 3, or SD3) and peak DUX4-FL expression at SD9. Overall, bi-transgenic mice without TMX (referred to as the mild FSHD-like model) exhibited very low