Stac3 is a novel regulator of skeletal muscle development in mice

Abstract

The goal of this study was to identify novel factors that mediate skeletal muscle development or function. We began the study by searching the gene expression databases for genes that have no known functions but are preferentially expressed in skeletal muscle. This search led to the identification of the Src homology three (SH3) domain and cysteine rich (C1) domain 3 (Stac3) gene. We experimentally confirmed that Stac3 mRNA was predominantly expressed in skeletal muscle. We determined if Stac3 plays a role in skeletal muscle development or function by generating Stac3 knockout mice. All Stac3 homozygous mutant mice were found dead at birth, were never seen move, and had a curved body and dropping forelimbs. These mice had marked abnormalities in skeletal muscles throughout the body, including central location of myonuclei, decreased number but increased cross-sectional area of myofibers, decreased number and size of myofibrils, disarrayed myofibrils, and streaming Z-lines. These phenotypes demonstrate that the Stac3 gene plays a critical role in skeletal muscle development and function in mice.

Figure 1. Skeletal muscle-predominant expression of Stac3 mRNA in mice.

Tissues from three adult C57BL/6 male mice were analyzed by real-time RT-PCR. In this analysis, 18s rRNA was used as an internal control. Data are expressed as mean ± SEM (n = 3). Bars not sharing the same letter labels are different (P<0.05). The y-axis is displayed on a log₁₀ scale. M: molecular ladder; SOL: soleus; EDL: extensor digitorum longus; TA: tibialis anterior; GAS: gastrocnemius.

Figure 2. Generation and analyses of Stac3 mutant mice.

A, Schematic representation of the targeted Stac3 allele. The wild-type (WT) Stac3 allele has 12 exons, with exon 3 containing the translation start codon. The targeted allele is inserted with a trapping cassette between exons 1 and 2, and this insertion is expected to disrupt the generation of normal Stac3 mRNA. Arrows indicate locations of PCR primers for genotyping. B, Genotypes and life of newborn offspring from intercrossing mice heterozygous for the targeted Stac3 allele. “+/+”, “+/−”, and “−/−” indicate wild-type mice, and mice heterozygous and homozygous for the targeted Stac3 allele, respectively. A total of 10 litters were included in this analysis. C, Images of a Stac3 homozygous mutant mouse and a wild-type littermate. Note the curved body shape and dropping forelimbs in the mutant. D, Genotyping by PCR. See panel A for the locations of two pairs of primers. E, Validation of diminished expression of Stac3 mRNA in Stac3 ^−/− mice by standard RT-PCR. Shown is a representative image. F, Real-time RT-PCR validation of diminished expression of Stac3 mRNA in Stac3 ^−/− mice. Data are expressed as mean ± SEM (n = 4). ** P<0.01 vs. “+/+”. G, LacZ staining of E13 wild-type and Stac3 ^+/− embryos. Shown are representative images. The image on the right highlights the stained somites in an E13 Stac3 ^+/− embryo.

Figure 3. Histological analyses of skeletal muscles in newborn Stac3 homozygous mutant mice (−/−) and wild-type (+/+) littermates.

A–H, Hematoxylin and eosin staining of diaphragm, tongue, tibialis anterior (TA), and extensor digitorum longus (EDL) muscles. Note the difference in the location of myonuclei between the two genotypes. I and J, Immunohistochemical staining of EDL muscle for myosin heavy chain protein (red) and nuclei (blue). Shown are representative images taken from matched areas. Scale bars  = 50 μm for micrographs A, B, I, and J. Scale bars  = 25 μm for micrographs C-H.

Figure 4. Quantitative analyses of extensor digitorum longus (EDL) muscle in newborn Stac3 ^−/− mice and wild-type and heterozygous littermates.

A, Percentage of myofibers that had centrally located nuclei. B, Total number of myosin heavy chain (MyHC)-positive myofibers at the widest girth of EDL muscle. C, Average cross-sectional area of myofibers in EDL muscle. D, Frequency distribution of cross-sectional areas (CSA) of myofibers in EDL muscle. Data are expressed as mean ± SEM (n = 3 to 7). * P<0.05 and ** P<0.01 vs. wild-type or heterozygous mice.

Figure 5. Representative electron micrographs of extensor digitorum longus (EDL) muscle of newborn Stac3 ^−/− mice and Stac3 ^+/+ littermates.

Note the differences in the location of myonuclei and the size and arrangement of myofibrils between Stac3 homozygous mutant and wild-type muscles. Arrows point to myonuclei (micrographs A and B), and arrowheads indicate Z-lines (micrographs C and D). Scale bars  = 10 µm for micrographs A and B; Scale bars  = 500 nm for micrographs C and D.