Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice

Abstract

Serum response factor (SRF) controls the transcription of muscle genes by recruiting a variety of partner proteins, including members of the myocardin family of transcriptional coactivators. Mice lacking SRF fail to form mesoderm and die before gastrulation, precluding an analysis of the roles of SRF in muscle tissues. To investigate the functions of SRF in skeletal muscle development, we conditionally deleted the Srf gene in mice by skeletal muscle-specific expression of Cre recombinase. In mice lacking skeletal muscle SRF expression, muscle fibers formed, but failed to undergo hypertrophic growth after birth. Consequently, mutant mice died during the perinatal period from severe skeletal muscle hypoplasia. The myopathic phenotype of these mutant mice resembled that of mice expressing a dominant negative mutant of a myocardin family member in skeletal muscle. These findings reveal an essential role for the partnership of SRF and myocardin-related transcription factors in the control of skeletal muscle growth and maturation in vivo.

Fig. 1.

Deletion of Srf with a skeletal muscle-specific Cre transgene. (A) Myo-Cre transgenic mice were bred with ROSA26R indicator mice to determine the temporal and tissue specificity of Cre expression. Whole-mount photographs of β-galactosidase-stained embryos of the indicated embryonic ages are shown. The lacZ reporter gene is activated specifically in the skeletal muscle lineage. (B) WT and Srf^flex1/flex1/Myo-Cre (KO) mice immediately after birth are shown. The mutant is cyanotic and displays curvature of the spine. (C) The structure of the Srf^flex1 allele before (Upper) and after (Lower) Cre-mediated recombination is shown. Triangles represent loxP sites. Exons 1 and 2 are shown in black boxes with the 5′ UTR as a white box. Primers used by PCR are designated L and R, and sizes of PCR fragments are indicated. (D) PCR of genomic DNA from skeletal muscle of mice of the indicated genotypes. Primers L and R yield a product of 1,340 bp with the Srf^flex1 allele and 380 bp with the Srf^/x1 allele in the presence of the Myo-Cre transgene.

Fig. 2.

Histology of skeletal muscle of Srf^flex1/flex1/Myo-Cre mice. (A) Histological sections of representative muscle groups of WT and Srf^flex1/flex1/Myo-Cre mice at E19.5 were stained with H&E. The muscle fibers in the mutant are thinner than those of WT. (Bar: 20 μm.) (B) Hindlimb muscle of WT and Srf^flex1/flex1/Myo-Cre mice was analyzed by electron microscopy at E19.5. The muscle fibers in the mutant are disorganized and less developed than those of WT. Magnifications are shown at left. (Bar: 2 μm, Upper; 0.5 μm Lower.)

Fig. 3.

Analysis of muscle markers in Srf^flex1/flex1/Myo-Cre mice. RNA was isolated from hindlimb muscles of WT and Srf^flex1/flex1/Myo-Cre (KO) mice at birth and analyzed by semiquantitative RT-PCR for the indicated transcripts. Samples from two animals of each genotype are shown.

Fig. 4.

Deletion of Srf with a MCK-Cre transgene. (A) WT and Srf^flex1/flex1/MCK-Cre (KO) mice at P3 are shown. The mutant is severely runted. (B) PCR of genomic DNA from mice of the indicated genotypes. Primers L and R yield a product of 1,340 bp with the floxed Srf^flex1 allele and 380 bp with the deleted Srf^lx allele, generated in the presence of the MCK-Cre transgene. (C) Histological sections of representative muscle groups of WT and Srf^flex1/flex1/MCK-Cre mice were stained with H&E. The cross-sectional area of the muscle fibers in the mutant is smaller than that of WT. (Bar: 20 μm.) (D) RNA was isolated from hindlimb muscles of WT and Srf^flex1/flex1/MCK-Cre (KO) mice at P3 and analyzed by semiquantitative RT-PCR for the indicated transcripts.

Fig. 5.

Skeletal muscle abnormalities resulting from expression of dnMRTF-A. (A) Western blot analysis of skeletal muscle from MCK-dnMRTF-A transgenic mice. Extracts from skeletal muscle of WT and MCK-dnMRTF-A transgenic mice were analyzed by Western blot with anti-FLAG antibody to detect FLAG-tagged dnMRTF-A. Two transgenic lines are shown. (B) Hindlimb muscles of WT and MCK-dnMRTF-A transgenic (line 1) mice at 4 weeks of age are shown. The transgenic animal shows severe skeletal myopathy. (C) Histological sections of hindlimb muscles of WT and MCK-dnMRTF-A transgenic mice at 4 weeks of age were stained with H&E. The muscle fibers in the transgenic animals are thinner than those of WT. Transgenic line 1 shows the most severe phenotype with extensive fibrosis and centrally located nuclei. (Bar: 20 μm.) (D) RNA was isolated from hindlimb muscles of WT and MCK-dnMRTF-A transgenic (Tg) mice line 1 at 4 weeks of age and analyzed by semiquantitative RT-PCR for the indicated transcripts.