(A–F) Transverse limb muscle sections of E9.5 control (A–C) and DMB-treated fetuses (D–F) were immunostained with YAP and MF20 antibodies and labeled with Hoechst to visualize nuclei in blue (N = 3). (A–C) In myofibers, YAP was preferentially localized in myonuclei (arrows). (D–F) In paralyzed limb muscles, the nuclear localization of YAP protein in muscle fibers (MF20+ cells in red) was lost (D–F, arrows) compared to control muscles (A–C, arrows). (G) Focus on YAP+ myonuclei in MF20+ cells in control muscles and YAP- myonuclei in paralyzed muscles (DMB). (H) Quantification of the percentage of YAP+ myonuclei in MF20+ cells in control and paralyzed muscles. In situ hybridization to YAP target genes, ANKRD1 (I,K,L,N,O) and CTGF (J,M,Q,R) followed by immunohistochemistry with MF20 antibody in control limbs (I-M, Q) and paralyzed muscles (N,O,P,R) (N = 4). (I–M, Q) The YAP target genes were expressed at the tips of muscle fibers (blue staining in MF20+ cells in brown) visualized on longitudinal (I–K,Q) and transverse (L,M) muscle sections. ANKRD1 was exclusively expressed in limb muscles (L,K), while CTGF (M,Q) displayed additional expression in cartilage. (N,O,P,R) In the absence of muscle contraction, the expression of ANKRD1 and CTGF was lost in muscles but not in cartilage for CTGF. u, ulna, r, radius. For transverse limb sections, dorsal is to the top and posterior to the left. For longitudinal sections, dorsal is to the top and proximal to the left. (S) RT-q-PCR analyses of the expression levels of YAP target genes in limbs of 12 hr and 48 hr DMB-treated embryos. For each gene, the mRNA levels of control limbs were normalized to 1. The graph shows means ± standard errors of the mean of nine biological replicates. The p-values were calculated using the Mann-Whitney test. Asterisks indicate the p-value, **p<0.01, ***p<0.001, ****p<0.0001.