Fig. 3.
Prox1 expression is enriched in slow-twitch skeletal muscle and is significantly reduced following skeletal-muscle–specific ablation. (A) Immunostaining of sections from wild-type and mutant slow-twitch soleus (sol) and fast-twitch EDL muscles. Prox1 is expressed in the nucleus (red) in slow- and fast-twitch skeletal muscle, identified by presence of type I and type IIb fibers, respectively (green). Nuclear Prox1 staining in wild-type muscles is highlighted by white arrowheads; residual Prox1 expression in mutant soleus muscle is highlighted by white arrows and is indicative of incomplete Myf5-Cre–induced knockdown. (Scale bar, 20 μm.) (B) Analysis of the proportion of Prox1-expressing nuclei indicates significant enrichment of Prox1 in slow-twitch muscle compared with fast-twitch muscle and significant knockdown of Prox1 in fast- and slow-twitch muscle following Myf5-Cre ablation (n = 3). (C) Quantitative RT-PCR for Prox1 transcripts in control (Co, Prox1fl/fl), cre (Cre, Myf5Cre/+;Prox1+/+), het (Het, Myf5Cre/+;Prox1fl/+), and mutant (Mut, Myf5Cre/+;Prox1fl/fl) animals reveal enrichment of Prox1 in slow-twitch muscle (green) and significantly reduced expression in fast- and slow-twitch mutant muscles compared with control. (D) Western blot analysis and (E) quantification of Prox1 expression in control (Co, Prox1fl/fl), het (Het, Myf5Cre/+;Prox1fl/+), and mutant (Mut, Myf5Cre/+;Prox1fl/fl) animals show enrichment of Prox1 in slow-twitch muscle and reduced expression in slow- and fast-twitch mutant muscles. (F) Expression of Tnnt3, Tnni2, and Myl1 in fast-twitch (red) and slow-twitch (green) muscles of control (Co, Prox1fl/fl), cre (Cre, Myf5Cre/+;Prox1+/+), het (Het, Myf5Cre/+;Prox1fl/+), and mutant (Mut, Myf5Cre/+;Prox1fl/fl) animals at 4 wk. Levels are expressed relative to control fast-twitch muscle (red) (n > 5). Significance was determined by Student t test in comparison with control fast- or slow-twitch muscle levels, where appropriate: *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.