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First published online 3 August 2006
doi: 10.1242/dev.02499

Development 133, 3473-3484 (2006)
Published by The Company of Biologists 2006
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Bone morphogenetic protein receptor 1A signaling is dispensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation

Changwon Park1,2, Kory Lavine3, Yuji Mishina4, Chu-Xia Deng5, David M. Ornitz3 and Kyunghee Choi1,2,*

1 Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA.
2 Developmental Biology Program, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA.
3 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA.
4 Molecular Developmental Biology Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
5 Genetics of Development and Disease Branch, NIDDK, NIH, Bethesda, MD 20892, USA.


Figure 1
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  		Fig. 1. Generation of Flk1+/CreAlk3floxed/floxed mice. (A) The mating scheme used to generate Alk3 CKO and control littermates. (B) (Upper) A 150 bp fragment from wild-type Alk3 allele and a 230 bp fragment from floxed Alk3 allele can be PCR-amplified with primers indicated by gray triangles (sense, 5'-GCAGCTGCTGCTGCAGCCTCC-3'; antisense, 5'-TGGCTACAATTTGTCTCATGC-3'). A 180 bp fragment should appear with primers indicated by black triangles (sense, 5'-GGTTTGGATCTTAACCTTAGG-3'; antisense, 5'-TGGCTACAATTTGTCTCATGC-3') if Cre-mediated recombination occurs. The presence of the knock-in Cre gene was confirmed by a 540 bp fragment using: sense, 5'-GAAACCGAGCTGCGTCCAGATTT-3'; antisense, 5'-GGTGTACGGTCAGTAAATTGG-3'. (Lower) A representative tail genomic DNA PCR results. Lane 1, Alk3+/floxed; lane 2, Flk1+/CreAlk3+/floxed; lane 3, Flk1+/CreAlk3floxed/floxed. (C-J) Gross morphology of Alk3 CKO embryos. Scale bars: 1 mm.

 

Figure 2
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  		Fig. 2. Hematopoietic potential of Alk3 CKO and Smad4 CKO embryos. E9.5 Alk3 CKO yolk sacs (A), E10.5 fetal liver (D), Smad4 CKO yolk sacs (E; upper, E8.5; lower, E9.5) were dissociated and plated into methylcellulose cultures with hematopoietic cytokines. Four to seven days later, colonies were counted and plotted. Bar indicates the average number of colonies; error bars indicate standard deviations; n, number of embryos. One representative result is shown. Similar results were obtained from three independent experiments. Ery, erythrocytes; Mac, macrophage, CFU-E, colony forming unit-erythrocyte; BFU-E, burst forming unit-erythrocyte. (B) Genomic PCR of sorted FLK1+ or TER119+ cells in E9.5 yolk sacs. Lane 1, molecular marker; lane 2, sorted cells from Flk1+/CreAlk3floxed/floxed; lane 3, sorted cells from Alk3floxed/floxed; lane 4; whole yolk sac cells from Alk3floxed/floxed; lane 5, whole yolk sac cells from Flk1+/CreAlk3+/floxed. (C) Genomic PCR on hematopoietic colonies grown from E9.5 Alk3 CKO yolk sac replating. Lane 1, Alk3floxed/floxed; lane 2, Flk1+/CreAlk3+/floxed; lane 3, Flk1+/CreAlk3floxed/floxed; lane 4, Alk3+/floxed; lane 5, positive control; lane 6, Negative control (H2O).

 

Figure 3
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  		Fig. 3. Vessel remodeling and maturation defect in Alk3 CKO embryos. Yolk sacs (A,B) and embryo proper (C,D) were subjected to whole-mount staining with anti-PECAM1 (CD31) antibodies and were visualized by alkaline phosphatase. The Alk3 CKO embryos display the primary vascular plexus in yolk sacs (B) and abnormal vascular patterning in the brain (D), but the controls showed normal vasculature pattern (A,C). Scale bars: 1 mm. (E) RNAs from E8.5-E10.5 yolk sacs were subjected to qRT-PCR for Id genes (Id1-Id4). The relative expression level of Id1, Id2, Id3 and Id4 in wild-type yolk sacs is shown. (F) Expression of all Id genes, Plau1 and Serpine1 was examined and compared in the Alk3 CKO and the controls. RNAs from two wild-type (wt) and two Alk3 CKO embryos were used for qRT-PCR, respectively. *P<0.05.

 

Figure 4
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  		Fig. 4. Disorganized vessel structure in the Alk3 CKO dorsal aorta. (A-D') Anti-PECAM (CD31) antibody staining. The Alk3 CKO embryos often exhibited dilated vessels (indicated by arrowheads) in the brain and abnormal patterns of vessel branching in the trunk (B,D,D'), compared with the controls (A,C,C'). (C',D') Higher magnification of the insets of C and D, respectively. Scale bars: 1 mm. (E-J) E10.5 embryos were stained with anti-PECAM1 (CD31) antibody (red, F,I) to visualize endothelial cells and anti-SMC {alpha}-actin antibody (green, G,J) to visualize SMCs. The endothelial cell layer of the dorsal aorta appeared to be normal, as indicated by anti-CD31 staining in both controls (wild-type) and Alk3 CKO embryos. SMCs are readily found throughout the dorsal aorta in the controls, but in the mutants SMCs are lacking in some areas of the dorsal aorta. Arrows (E,H) indicate blood cells in the lumen of the dorsal aorta. Three wild-type and three Alk3 CKO embryos were analyzed in cross-section. Scale bars: 50 µm. (K) Quantitative RT-PCR analysis of dorsal aorta for Cd31, SMC {alpha}-actin, Mhc, Angpt1, Tie2, Vegf, Pten, integrin {alpha}v and integrin ß3. Three wild-type and three Alk3 CKO embryos were used for qRT-PCR. *P<0.01. (L) E10.5 embryos were sectioned and examined under transmission electron microscopy. There are breaks in the Alk3 CKO dorsal aorta (red arrowheads). In addition, the mutant endothelial cell layer did not form a close association with SMCs, compared with the controls. Two control embryos and four Alk3 CKO embryos were analyzed for EM study. SMC, smooth muscle cells; Endo, endothelial cells. Scale bars: 6 µm.

 

Figure 5
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  		Fig. 5. Defects in vessel remodeling and the heart of the Smad4 CKO embryos. (A) E9.5 and E10.5 yolk sacs of Smad4 CKO were subjected to whole-mount staining with anti-PECAM1 (CD31) antibody. The controls (wild type) showed complex branching (left), but Smad4 CKO embryos displayed only the primary vascular plexus (right). Scale bars: 500 µm. (B) Expression of Id genes in the Smad4 CKO yolk sacs. RNAs from two wild-type and three Smad4 CKO embryos were used for qRT-PCR. *P<0.05. (C-D') Hematoxylin and Eosin staining of E9.5 heart of Smad4 CKO embryos. (C'-D'') Higher magnification of the insets (green and black) in C and D, respectively. Arrows indicate the cushion in the AVC and arrowheads indicate the trabeculae in the ventricle. Scale bars: 200 µm.

 

Figure 6
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  		Fig. 6. The Alk3 CKO embryos exhibit defects in the cushion formation in the AVC, but not in the OFT. (A) E10.5 wild-type (n=4) and Alk3 CKO (n=4) embryos were sectioned sagittally and stained with Hematoxylin and Eosin. In the control (wild-type) embryos, trabeculae and endocardial cushions in the AVC region, as well as the OFT develop normally. The AVC cushion is missing in the Alk3 CKO embryos, but the development of the OFT cushion persists. Black arrows and white arrows represent the cushion in the AVC and in the OFT, respectively. Middle and bottom panels represent higher magnification of the AVC and the OFT, respectively. Scale bars: 200 µm. (B) Flk1+/Cre mice were crossed with Rosa26R-lacZ mice and the resulting embryonic hearts were cross-sectioned, and subjected to lacZ staining. At E10.5, the majority of lacZ-positive cells in the AVC are present in the cushion cell layer and in the endocardium. However, in the OFT, the lacZ-positive cells are limited to the endocardium. By E12.5, all ACV cushion cells are lacZ+, while the OFT cushion consists of both lacZ+ and lacZ- cells. (C) Alcian Blue staining. (D) Quantitative RT-PCR analysis of E10.5 heart lacking the OFT and right ventricle for Snai1, VE-Cadherin (VE-Cad), Sox9, Nfatc1, Tgfb2, Msx1 and Twist1. Four wild-type and four mutant embryos were used for quantitative RT-PCR analysis. *P<0.05.

 

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© The Company of Biologists Ltd 2006