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First published online 22 October 2003
doi: 10.1242/dev.00831

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Embryonic atrial function is essential for mouse embryogenesis, cardiac morphogenesis and angiogenesis

Institute of Molecular Medicine and Department of Medicine, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0641, USA

View larger version (28K): [in a new window]   Fig. 1. Targeted generation of MLC2a-null mice. (A) Targeting strategy. A restriction map of the relevant genomic region of MLC2a (top), the targeting vector for MLC2a (middle) and the mutated/targeted locus after recombination (bottom) are shown. The translational start site is indicated by ATG. Orientations of Cre and neomycin resistance genes (Neo) are indicated by arrows. A, Acc1; B, BamH1; H, HindIII; K, Kpn1; S, Sst1; X, Xba1. (B) Detection of wild-type and targeted alleles by Southern blot analysis. DNA from electroporated MLC2a ES cells was digested with Sst1 and analyzed by Southern blot analysis with a probe as shown in A. The 9.5 and 7.5 kb bands represent wild-type and targeted alleles, respectively. (C) Detection of MLC2a by protein analysis. Proteins were prepared from E9.5 MLC2a-null (–/–) and wild-type (+/+) embryos, and analyzed with MLC2a antibodies.

View larger version (76K): [in a new window]   Fig. 2. Microscopic and histological assessment of physical defects in MLC2a-null embryos. Whole embryo assessment of wild-type (A) and MLC2a-null (G) mouse embryos at somite pairs 24. Note that the null mutant displays severe chest edema. Paraffin wax-embedded serial sections of wild-type (B-F) and MLC2a-null (H-L) mouse embryos were stained for nuclei and cytoplasm with Hematoxylin and Eosin, respectively. White arrows point out the lack of mesenchymal cell seeding in atrioventricular cushions of MLC2a mutant (K), in contrast to normal mesenchymal cell seeding in atrioventricular cushions of wild-type control (E). Black arrows in D and J indicate dilated ventricle. A, atria; OT, outflow tract; RA, right ventricle; LA, left atria; RV, right ventricle; LV, left ventricle; SV, sinus venosus.

View larger version (84K): [in a new window]   Fig. 3. Assessment of cardiac defects in MLC2a-null embryos. Left (A,D,G,J,M,P,S,V), front (B,E,H,K,N,Q,T,W) and right (C,F,I,L,O,R,U,X) views of wild-type (+/+) and MLC2a-null (–/–) embryos at somite pairs 10 (A-F), 13 (G-L), 16 (M-R) and 22 (S-X). Relative to the linear heart tubes of wild-type littermates (A-C), mutant heart tubes appeared to be enlarged and amorphous, with no clear distinction between the bulbus arteriosus and future left ventricle of the heart (D-F). With progression through looping morphogenesis, mutant hearts exhibited aberrant morphologies in each cardiac chamber, accompanied by overall abnormalities in looping architecture (J-L,P-R,V-X).

View larger version (182K): [in a new window]   Fig. 4. Ultrastructural analysis of atrial and ventricular myocytes from MLC2a-null and wild-type somite matched embryos. Representative images of cardiac atria and left ventricles of wild-type (+/+) and MLC2a-null (–/–) embryos at sp 22, as assessed by transmission electron microscopy. Black arrowhead indicates the lack of myofibrillar organization in atrial sections from MLC2a-null mutants (C), in contrast to the organized myofibrillar structure observed in atrial sections from wild-type littermates (A). White arrows indicate the normal Z line formation seen in left ventricular sections from both wild-type (B) and MLC2a-null (D) embryos.

View larger version (18K): [in a new window]   Fig. 5. Baseline atrial and ventricular heart rates of MLC2a-null and wild-type littermate embryos. Mean heart rates at baseline are shown for both cardiac atria and ventricles of wild-type (WT) and MLC2a-null (KO) mouse embryos at ED 9.5. Mean heart rates, measured in beats per minute (bpm), were 51±6 and 4±4 for WT and KO atria, and 53±10 and 48±7 for WT and KO ventricles, respectively.

View larger version (62K): [in a new window]   Fig. 6. Microscopic and histological assessment of extra- and intraembryonic vascular formation in MLC2a-null mutants and wild-type littermates. (A-I) Whole-mount assessment of wild-type (+/+) and MLC2a-null (–/–) yolk sacs at ED8.5 (A,F), ED9 (B,G), ED9.5 (C,H) and ED10 (D,I). (E,J) Paraffin wax-embedded sections from wild-type (E) and MLC2a-null (J) yolk sacs at ED10 were stained for nuclei and cytoplasm with Hematoxylin and Eosin, respectively. Separation of mesodermal and endodermal layers was observed in the MLC2a-null yolk sac (J). (K-P) Whole-mount immunostaining of wild-type (+/+) and MLC2a-null (–/–) yolk sacs for Flk1 at ED8.5 (K,N), and PECAM at ED9 (L,O) and ED9.5 (M,P). (Q-T) Whole-mount immunostaining of wild-type (Q,R) and MLC2a-null (S,T) embryos for PECAM at somite pair 24. White arrows point out the lack of organization of cranial and intersegmental vessels in MLC2a-null embryos, relative to controls.

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