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First published online 30 July 2008
doi: 10.1242/dev.017863

Development 135, 2981-2991 (2008)
Published by The Company of Biologists 2008
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SM22{alpha}-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis

Nesrine El-Bizri1,2, Christophe Guignabert1,2, Lingli Wang1,2, Alexander Cheng1,2, Kryn Stankunas3, Ching-Pin Chang3, Yuji Mishina4 and Marlene Rabinovitch1,2,*

1 Cardiopulmonary Research Program, Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University School of Medicine, Stanford, California, CA XXXXX?, USA.
2 Department of Pediatrics, Stanford University School of Medicine, Stanford, California, CA XXXXX?, USA.
3 Department of Medicine, Stanford University School of Medicine, Stanford, California, CA XXXXX?, USA.
4 Molecular Developmental Biology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, NC XXXXX?, USA.


Figure 1
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  		Fig. 1. Genotyping and phenotyping SM22{alpha}-Cre;Bmpr1aflox/flox mouse embryos. (A) Genotyping of mouse embryos. Targeted deletion of exon 2 of Bmpr1a occurs only in mice expressing Cre and the floxed Bmpr1a gene. SM22{alpha}-Cre;Bmpr1aflox/+ (f/+) and flox/flox (f/f) represent mice heterozygous and homozygous for the floxed gene, respectively. Mice expressing either Cre or the floxed Bmpr1a gene represent the WT group. Any mutant (f/fR26R) or WT (WTR26R) mouse expressing Cre and the R26R gene, expresses β-galactosidase and can be used for lacZ staining. (B) Cre activity in SM22{alpha}-Cre;R26R;Bmpr1aflox/flox embryos. Whole-mount lacZ staining of SM22{alpha}-Cre;R26R;Bmpr1aflox/+ embryos showing blue staining (a) in the heart (asterisk) and dorsal aorta (arrow) at E9.25, and (b) in smaller vessels as well as somatic myotomes (arrows) at E10.5. (C) Phenotype of SM22{alpha}-Cre;R26R;Bmpr1aflox/flox (b,d,f) compared with WT (a,c,e) embryos. SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mutants appear normal at E9.5 (b versus a) and relatively reduced in size at E10.5 (d versus c); at E11, areas of hemorrhage are noted in the heart (asterisk) and abdomen (asterisk), as well as near the mouth (arrow) and brain (arrowhead) (f versus e). (D) Percentage of total mice of SM22{alpha}-Cre;R26R;Bmpr1aflox/flox genotype. Numbers of genotyped mice of each age are depicted in parentheses.

 

Figure 2
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  		Fig. 2. Cardiac defect in SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Blue staining (Cre activity) in the atrial (am) and ventricular (vm) myocytes but not in endocardial cells (ec) by whole-mount lacZ staining in E10.5 SM22{alpha}-Cre;R26R;Bmpr1aflox/flox embryos. (B) An enlargement of A. (C,D,F,G,H,I) Consecutive transverse sections of wild-type (WT) (C,F,H) and SM22{alpha}-Cre;R26R;Bmpr1aflox/flox (D,G,I) hearts taken at the same level from viable E11 embryos. H&E staining shows thinning of the ventricular wall (arrowheads in high-magnification insets) in the flox/flox mutant heart (D) versus WT (C). Apoptosis was infrequent in ventricular sections of E11 SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mutant (G) and WT (F) by TUNEL immunostaining. However, fewer PCNA-positive cells (brown) were observed in the mutant ventricles (I, high-magnification inset, arrows) compared with the WT (H, inset, arrows). (E) A numerical assessment of hematoxylin-stained nuclei per ventricular section of E10.5-11 WT and SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mutants. Bars indicate mean±s.e.m. (n=3). *P<0.05. (J) A numerical assessment of percentage of PCNA-positive cells over the total number of ventricular cells in heart sections of WT and flox/flox (f/f) mutant embryos at E9.5 and E10.5-11. Bars indicate mean±s.e.m. (n=3-4). *P<0.05. (K,L) p57KIP2 immunostaining in E10.5 WT (K) and flox/flox mutant (L) heart sections. RA, right atrium; RV and LV, right and left ventricle, respectively; EC, endocardial cushions; IVS, interventricular septum. Panels depicting WT and their corresponding mutants have the same magnification. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. Dilated large vessels with reduced VSMCs in SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Whole-mount PECAM staining of E10.5 embryos (a-f) shows a dilated aorta in the SM22{alpha}-Cre;R26R;Bmpr1aflox/flox compared with WT (d versus c, arrowheads). Panels c and d are high magnifications of the framed areas in a and b, respectively. Dilatation of large abdominal vessels with frequent interconnections are seen in the flox/flox mutants versus WT (f versus e, arrowheads). H&E-stained transverse sections show dilatation of the mutant aortae (h) compared with WT (g). Poor investment of lacZ staining (blue) SM22{alpha}-expressing VSMCs around the mutant aortae in sections of whole-mount mutant embyros (j) versus WT (i). Sections h and j are at the same level as g and i, respectively. (B) {alpha}SM-actin staining (brown) shows poor investment of SMCs in the dilated aortic wall of SM22{alpha}-Cre;R26R;Bmpr1aflox/flox (b) versus WT (a). TUNEL staining (arrows) revealed similar occasional apoptotic cells surrounding the dilated aorta in the flox/flox mutant (d) and WT (c). However, reduced PCNA staining (arrows) was seen in VSMCs of the dilated aorta and in the neighboring mesenchymal cells in the flox/flox (f) versus WT (e). Panels a, c and e are similar consecutive sections in the WT embryo as b, d and f in the flox/flox mutant embryo. (g) Quantification of percentage of PCNA-positive cells over total number of SMCs in the vessel walls in dorsal aortae of E10.5-11 WT and SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mutants. Bars indicate mean±s.e.m. (n=4). *P<0.05. Panels depicting WT and mutants are of the same magnification. Scale bars: 100 µm in Ah,j; 50 µm in Bf.

 

Figure 4
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  		Fig. 4. Brain distortion in SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Whole-mount lacZ staining of an E10.5 WT embryo brain showing blue staining in the forebrain. (B) Collapse of telencephalic vesicles (b arrows) and compressed brains (d) of SM22{alpha}-Cre;R26R;Bmpr1aflox/flox versus WT (a,c) embryos at E10.5. H&E-stained cross-sections of WT (e,g,i,k) and mutant (f,h,j,l) head at the levels of the dashed lines in c and d, respectively. Sections 1, 2 and 3 are at levels of the frontonasal processes and section 4 is at the nasal-mandibular level. Panels depicting WT and mutants are of the same magnification. (C) Whole-mount PECAM staining shows unilateral clearing of telancephalic vessels in the WT (a,c) but not the flox/flox (b,d) head at E10.5. The clearing is evident at nasal-mandibular areas of the WT head (e) as opposed to resistance to clearing (arrows) in the mutant (f). TUNEL staining (arrows) on brain sections show less apoptosis in the mutants (h) than WT (g). By TUNEL assay (red) combined with NG2 immunofluorescence (green), no apoptosis was seen in NG2-positive areas in the WT brain sections (i); however, more immunoreactivity was seen in the mutants (j). Panels depicting WT are of the same magnification as their corresponding mutants. Percentage TUNEL-positive (k) and PCNA-positive (l) over total number of cells in nasal-mandibular areas (examined using a 20x objective) of E10.5 WT and SM22{alpha}-Cre;R26R; Bmpr1aflox/flox (f/f) mutants. Bars indicate mean±s.e.m. (n=3 in k and n=3-4 in l). *P<0.05. Scale bars: 100 µm in Cf,h,j; 200 µm in Bl.

 

Figure 5
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  		Fig. 5. Attenuated MMP9 and MMP2 expression in SM22{alpha}-Cre;R26R;Bmpr1aflox/flox mouse embryos. (A) Profiling by qRT-PCR the expression of genes that might be modified by the SM22{alpha}-targeted deletion of Bmpr1a. Values are shown relative to 18S RNA. n=3 where each sample combines 3-4 embryos. *P<0.05. Eln, elastin; Colla1, collagen Ia1. (B) Reduction of MMP9 (b versus WT in a) and MMP2 (d versus WT in c) protein expression in aortic walls of flox/flox embryos as assessed by immunofluorescence. Note the absence of immunoreactivity in an aortic section of a WT incubated with only the secondary antibody as a negative control (e). Panels depict WT and mutants at the same magnification. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. RNAi-induced loss of BMPR1A, by reducing MMP9 and MMP2 expression, attenuates proliferation of human vascular smooth muscle cells (HPASMCs). (A) Gelatin zymography using conditioned media of HPASMCs. Gelatin zymograms performed on conditioned media (left) and densitometric analysis (right) show MMP9 and MMP2 activities in SiBMPR1A-treated (SiBR1A) versus SiControl-treated (SiC) cells in response to serum (10% FBS) for 6 hours. Bars indicate mean±s.e.m. of densitometric values of SiBR1A proMMP9, MMP9, proMMP2 and MMP2 normalized to their corresponding SiC values (n=3-4). *P<0.05 and **P<0.01 between SiBR1A and SiC. (B) Proliferation of HPASMCs in response to serum using the MTT assay. HPASMCs transfected with SiC (white bars), SiBR1A, SiMMP9, SiMMP2 and combined SiMMP9 and SiMMP2 were subjected to 0.1% FBS (all white bars) or stimulated with 10% FBS (all black bars) for 72 hours, after which proliferation was assessed by the MTT assay. Bars indicate mean±s.e.m. of arbitrary OD570 values normalized to values of SiC under 0.1% FBS. n=12 for siMMP2 and/or siMMP9 and n=26 for SiC and SiBMPR1A from three independent experiments. {dagger}{dagger}{dagger}P<0.001 for serum-stimulated versus unstimulated comparisons, and ***P<0.001 for comparisons with SiC at 10% FBS.

 

Figure 7
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  		Fig. 7. RNAi-induced loss of BMPR1A, by reducing MMP9 and MMP2 expression, impairs directed migration of human vascular smooth muscle cells. (A) Gelatin zymography in response to PDGF-BB. Gelatin zymography performed on conditioned media (left) and densitometric analysis (right) show MMP9 and pro and active MMP2 activities in SiBMPR1A-treated (SiBR1A) versus SiControl-treated (SiC) cells in response to PDGF-BB (20 ng/ml) stimulation for 6 hours. Bars indicate mean±s.e.m. of densitometric values of SiBR1A MMP forms normalized to the corresponding SiC values. (n=4). **P<0.01, ***P<0.001. (B) Migration in response to PDGF-BB as assessed by modified Boyden Chamber assay. SiC- or SiBR1A-transfected HPASMCs were stimulated with 20 ng/ml of PDGF-BB for 6 hours. Bars represent mean±s.e.m. of migrating cells in 5-6 different microscopic fields. (n=4). {dagger}P<0.05 for stimulated versus unstimulated comparisons for each Si; ***P<0.001 for comparisons between SiBR1A and SiC. Representative micrographs show the number of migrating cells under each condition. Scale bar: 100 µm.

 

Figure 8
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  		Fig. 8. RNAi-induced loss of BMPR1A, by reducing MMP2 expression, attenuates apoptosis of human pericytes (HBVPs). (A) Gelatin zymography using conditioned media of HBVPs in response to serum deprivation. Gelatin zymograms of conditioned media (top) with densitometric analysis (beneath) to assess MMP2 activities in SiBMPR1A-transfected (SiBR1A) as compared with SiControl-transfected (SiC) cells. Bars represent mean±s.e.m. of densitometric values of SiBR1A MMP2 forms normalized to the corresponding SiC value. n=7-8 for pro and n=3-4 for active MMP2. *P<0.05 versus SiC. (B) Reduced MMP2 activity in HBVPs transfected with MMP2 RNAi. Gelatin zymogram of conditioned media (top) and densitometric values (beneath) of MMP2 in HBVPs transfected with SiMMP2. Bars represent mean±s.e.m. of densitometric values of SiBR1A MMP2 forms normalized to SiC value (n=4). ***P<0.001. (C) Apoptosis of HBVPs in response to serum deprivation using caspase 3/7 assay. Apoptosis in HBVPs transfected with SiC, SiBR1A or SiMMP2 was induced by serum-deprivation for 24 hours and assessed by a caspase 3/7 luminescent assay. Bars represent mean±s.e.m. of arbitrary luminescent values (n=6-9). ***P<0.001 versus SiC.

 

Figure 9
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  		Fig. 9. Schematic of defective BMPR1A signaling in vascular smooth muscle cells and pericytes. Deletion of BMPR1A results in decreased MMP9 and MMP2 activities in VSMCs, and decreased MMP2 activity in pericytes, culminating in reduced proliferation and apoptosis, respectively.

 

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