SM22α-Cre activity in the R26R mouse. Mice carrying both the R26R and SM22α-Cre alleles stained for lacZ at e8.5 (A) and e10.5 (B–D). The blue staining in the indicated structures reflects β-galactosidase activity due to Cre-mediated excision of a floxed PGK neo cassette upstream of the lacZ gene within the ROSA26 locus (12). am, amnion; ao, aorta; at, atrium; he, heart; hf, head folds; v, ventricle; u, umbilical vessels.

Electron microscopy of e10.5 hearts. (A) Wild-type cardiac sarcomeres show periodic units of myofilaments delineated by Z disks (×10,000). (B) Higher magnification of boxed area in A demonstrating normal parallel arrangement of myofilaments and normal Z disk structure (×30,000). (C) Mutant cardiac sarcomeres with marked disorganization of myofilaments and distorted Z disks (asterisk) (×15,000). (D) Higher magnification of boxed area in C demonstrating an abnormal sarcomere with disorganized myofilaments and aberrant Z disks (×30,000). (E) Wild-type dorsal aortic SMC (arrowhead) immediately subjacent to endothelium (arrow) (×4,000). lu, lumen. (F) Higher magnification of boxed area in E illustrating a zone of actin/intermediate filaments (dashed circle). n, nucleus. These zones of thin filaments are free of any organelles and are often found near the plasma membrane of SMC (×20,000). Mutant SMC did not contain such zones of filament bundles (see text).

ISH and immunohistochemistry of cardiac/SMC markers. e10.5 (A, B, E, F, I, J, M, and N) and e11.5 (C, D, G, H, K, L, O, and P) embryos assayed for cardiac α actin (A–D), SM22α (E–H), SMA (I–L) mRNA, or SMA protein (M–P) are shown. Wild-type embryos (A, C, E, G, I, K, M, and O) show expression of markers in heart and aorta. Mutants at e10.5 show reduced transcripts to cardiac α actin (B), SM22α (F), and SMA (J), which are all absent at e11.5 (D, H, and L). Note persistent expression of SMA protein in heart and aorta of mutant at e11.5 (P). Small arrows indicate heart, and larger arrows indicate aorta.

Floxed SRF targeting allele. (A) Schematic of SRF targeting allele and position of loxP sites upstream of promoter and downstream of exon 1. Regulatory elements in proximal promoter are indicated. (B) PCR genotyping depicting wild-type allele vs. heterozygous and homozygous floxed alleles.

Cardiac/vascular morphology and SRF expression in wild-type vs. mutant embryos. Hematoxylin/eosin-stained wild-type (A) and mutant (B) e10.5 embryos are shown. (Bars, 400 μm.) SRF immunostaining of wild-type (C and E) and mutant (D and F) hearts shows marked differences in levels of SRF (brown nuclei) as well as cardiac trabeculation and compact zone thickness. (Bars, 30 μm.) E and F represent magnified images of the boxes in C and D, respectively. (Bars, 10 μm.) The dorsal aorta of wild-type mice shows greater accumulation of SRF-stained aortic SMC (G) than in mutants (H). (Bars, 10 μm.) Arrows indicate SRF positive cardiomyocytes (E) or SMC (G) in wild types, and arrowheads point to SRF-negative cardiomyocytes (F) and SMC (H) in mutants. I shows the percent SRF immunoreactivity in ventricular cardiomyocytes; n = 3–5 e10.5 embryos per time point. Beginning at e9.5, the difference between wild-type (black bars) and mutant (blue bars) cardiomyocyte SRF expression was statistically significant (asterisks here and below denote P < 0.05). J summarizes similarly quantitated SRF immunoreactive staining in subjacent SMC of the dorsal aorta; n = 3 e10.5 embryos per condition. K shows the number of SMC immediately subjacent to the endothelium per 100 μm of dorsal aorta; n = 3 e10.5 embryos per condition.

RNA expression of cardiac/SMC markers. Semiquantitative RT-PCR analysis was performed on total RNA from individual hearts (n = 3) of e10.75 wild-type (WT) or mutant (Mut) embryos. Each lane represents 28 cycles of PCR from one individual heart sample demonstrated to be within the linear range of amplification (data not shown). α-³²P-labeled PCR products were quantitated (see Materials and Methods), and the fold change is given as the mean of three measurements ± SD. ANF, atrial natriuretic factor; α MHC, α myosin heavy chain; β MHC, β myosin heavy chain; CAA, cardiac α actin; SKA, skeletal α actin, CNN1, SM calponin; NCX1, cardiac sodium–calcium exchanger; MYCD, myocardin.