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First published online 18 October 2006
doi: 10.1242/dev.02597

Development 133, 4585-4593 (2006)
Published by The Company of Biologists 2006
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Tgfß signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development

Kai Jiao1,2,3, Melissa Langworthy1,2, Lorene Batts1, Chris B. Brown1,4, Harold L. Moses5 and H. Scott Baldwin1,2,*

1 Division of Pediatric Cardiology, Department of Pediatrics, Vanderbilt Children's Hospital
2 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
3 Division of Genetic and Translational Medicine, Department of Genetics, The University of Alabama at Birmingham, Kaul 768, 720 20th Street S., Birmingham, AL 35294, USA.
4 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
5 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.


Figure 1
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  		Fig. 1. Myocardium-specific inactivation of Tgfbr2. (A,B) Whole-mount (A) and sectional (B) examination of a cTnTcre;Tgfbr2loxp/loxp;R26R embryo at E10.5 stained with X-gal. (C,D) PCR analysis of DNA isolated from either the myocardium (M) or endocardium (E) of the AVC region of an E10.0 cTnTcre;Tgfbr2loxp/loxp;R26R embryonic heart. (C) Primers p1 and p2 detected the unrecombined Tgfbr2loxp allele, whereas primers p1 and p3 detected the recombined allele (Bhowmick et al., 2004Go). (D) The recombined allele can be detected only from myocardial cells (lane 1) and not endocardial cells (lane 2), whereas the unrecombined allele is hardly detected from myocardial cells (lane 3 versus 4). The lacZ primers were used as a control to show that a similar amount of DNA template was used for the myocardium (lane 5) and endocardium (lane 6) in the PCR analysis. (E-H) Frontal sections of a wild-type (E) and a cTnTcre;Tgfbr2loxp/loxp (F-H) embryonic heart. The arrow in F indicates the VSD. Both the aorta and pulmonary trunk are connected to the right ventricle (G,H), resulting in a DORV defect. A, atrium; ao, aorta; cko, cTnTcre;Tgfbr2loxp/loxp; ctrl, wild-type embryos; E, endocardial cells; H, heart; LA, left atrium; LV, left ventricle; M, myocardial cells; oft, outflow tract; pt, pulmonary trunk; RA, right atrium; RV, right ventricle; V, ventricle.

 

Figure 2
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  		Fig. 2. Defective vasculogenesis in the yolk sacs of Tie2cre;Tgfbr2loxp/loxp;R26R embryos. (A,B) Embryos with yolk sacs were isolated at E10.5 (A) and E11.5 (B). The Tie2cre;Tgfbr2loxp/loxp;R26R embryos were arrested at around the 20-25 somite stage. No blood vessels were visible in their yolk sacs. (C) Embryos from A stained with X-gal to detect endothelial cells. In contrast to the well-formed blood vessel nets in the control embryo yolk sac, formation of blood vessels in the mutant was initiated only in the region close to the allantois (arrow). (D,E) Mutant embryo (E) that escaped global vascular compromise and survived to E12.5 without growth retardation or obvious yolk sac vascular abnormalities. cko, Tie2cre;Tgfbr2loxp/loxp;R26R embryos; ctrl, Tie2cre;Tgfbr2loxp/+;R26R embryos.

 

Figure 3
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  		Fig. 3. Tgfbr2 is not required for AV cushion mesenchyme formation. (A) The conditional knockout embryo (right) isolated at E10.5 was grossly normal compared with the control littermate (left). Embryos were stained with X-gal. The Tie2cre-mediated recombination in the mutant embryo occurred at the same level and with the same pattern as that in the control embryo (as judged from the R26R locus). (B) Mutant embryo isolated at E9.0, immediately before the onset of EMT in the AVC region, stained with X-gal and sectioned. (C-F) Control (C,E) and mutant (D,F) embryos isolated at E9.5 (C,D) and E10.5 (E,F), stained with X-gal and sagittally sectioned. The AV cushion mesenchymal cells formed normally in mutant embryonic hearts. (G) Count of the number of AV cushion mesenchymal cells in AVC regions of mutant and control embryonic hearts (E9.5). Data were averaged from four embryos of each genotype. Error bars indicate the s.d. (H) Sagitally sectioned mutant embryo isolated at E10.5, in which growth was arrested at around the 25 somite stage. Mesenchymal cells (arrowheads) can be observed in the AV cushions. (I) Quantitative analysis of the recombined Tgfbr2loxp allele amplified by quantitative real-time PCR analysis from genomic DNA isolated from AV endocardial/mesenchymal cells of control or mutant embryonic hearts (Fig. 1C). Lanes 1-3 represent a positive control in which a plasmid containing the recombined Tgfbr2loxp allele was used as a template, the copy number of which ranged from 104 to 102. Lane 4 is a no template control. Lanes 5-7 and lanes 8-10 represent the mutant and control samples, respectively, from three independent experiments. (J) The PCR product of the recombined Tgfbr2loxp allele could only be detected in the mutant samples. (K) Semi-quantitative RT-PCR analysis was performed with RNA isolated from the AV endocardial/mesenchymal cells. Lanes 1-3 are control samples with 100, 5 and 10% of input RNA. Lane 4 is the mutant sample with 100% of input RNA. GAPDH was used as a loading control. A, atrium; cko, Tie2cre;Tgfbr2loxp/loxp;R26R; ctrl, Tie2cre;Tgfbr2loxp/+;R26R; IC, inferior cushion; SC, superior cushion; V, ventricle.

 

Figure 4
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  		Fig. 4. Inactivation of Tgfbr2 in endocardial cells prevents EMT in vitro. (A,B) In vitro collagen gel assays performed with AV explants isolated from control (A) or mutant (B) embryonic hearts at E9.25. (C) The number of mesenchymal cells formed in control (Tgfbr2loxp/loxp;R26R) explants (n=18) is dramatically higher than that in mutant explants (n=14). *P<0.005. (D-G) Control (D,F) and mutant (E,G) explant cultures stained with an antibody (green) against the panendothelium marker Pecam (D,E), or against the mesenchyme marker {alpha}-smooth-muscle actin (F,G). Nuclei were visualized by staining with propidiumiodide (red). In the control, the mesenchymal cells expressed {alpha}-smooth-muscle actin (F) and lost the expression of Pecam (D). By contrast, the endocardial cells in mutant explant cultures expand on the gel surface with the expression of Pecam (E) but not of {alpha}-smooth-muscle actin (G). cko, Tie2cre;Tgfbr2loxp/loxp;R26R embryos; ctrl, control embryos.

 

Figure 5
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  		Fig. 5. Endocardial inactivation of Tgfß signaling causes DILV. (A,B) Whole-mount examination of E12.5 embryonic hearts from a control (A) and a mutant (B) embryo stained with X-gal. Red arrows show the interventricular sulcus at the apex of the heart. (C-H) Frontal sections of E12.5 hearts from control (C,F) or mutant (D,E,G,H) embryos stained with X-gal. Asterisks in C and D indicate the mesenchymal cap of the ASP. E represents the dark field view of D. Owing to penetration problems, which are common in X-gal staining studies, results of X-gal staining of E12.5 embryonic hearts varied dramatically among samples. X-gal staining products appear pink in the dark field, and can be detected more easily than in the bright field. Arrows in G indicate that both the right atrium (RA) and left atrium (LA) empty through separated `inlets' to the left ventricle, resulting in a DILV defect. (I) The relative cushion sizes of mutant embryonic hearts versus control hearts were measured as described in the Materials and methods. #P<0.05. (J-L) Cross sections of E12.5 hearts from control (J) and mutant (K,L) embryos show normal growth of the right ventricle. The section in K is close to the apex of the heart, and the section in L is at the level at which the VSD (black arrow) is obvious. (M) The size of the right ventricle is approximately the same as the left ventricle in both control and mutant embryos. (N-Q) Section in situ hybridization analysis performed on sagittally sectioned embryos (E9.5-E11.5) with a [35S]-labeled antisense probe (N-P) corresponding to the first four exons of Tgfbr2, or a sense probe as a negative control (Q). ASP, atrial septum premium; cko, Tie2cre;Tgfbr2loxp/loxp;R26R; ctrl, Tie2cre;Tgfbr2loxp/+;R26R; IC, inferior endocardial cushion; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; SC, superior endocardial cushion.

 

Figure 6
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  		Fig. 6. Cell proliferation study of AV cushions. (A-H) Frontal sections of control (A,B,E,F) and mutant (C,D,G,H) embryonic hearts at E11.5 stained with DAPI to visualize all nuclei (A,C,E,G) and with anti-Ki67 antibody to identify proliferating cells (B,D,F,H). A-D and E-H show the inferior and superior AV cushions, respectively. (I) The number of Ki67-positive nuclei as a percentage of total nuclei. Data (mean±s.d.) were collected from four embryonic hearts of each strain, and at least 500 nuclei were counted for each heart. (J-O) Frontal sections of control (J-L) and mutant (M-O) embryonic hearts (E11.5) stained with propidiumiodide (J,M) and an anti-cyclin D1 antibody (K,N). Only results from inferior cushions are shown. Identical confocal microscopic conditions were used to acquire the images of control and mutant samples. (L) Merged images of control (J,K) samples. (O) Merged images of mutant (M,N) samples. Arrows in M-O indicate examples of nuclei without cyclin D1. (P) Relative intensity of cyclin D1 immunostaining in mutant versus control hearts. The total intensity of cyclin D1 antibody staining (green) was determined using MetaMorph 5.0, and was averaged with the number of nuclei to acquire the average intensity. The intensity of the control superior cushion was set as 100%. (Q) The number of cyclin D1-positive nuclei as a percentage of total nuclei. For P and Q, data (mean±s.d.) were collected from five embryonic hearts of each strain, and at least 500 nuclei were counted for each heart. *P<0.05. cko, Tie2cre;Tgfbr2loxp/loxp;R26R; ctrl, Tie2cre;Tgfbr2loxp/+;R26R; IC, inferior cushion; SC, superior cushion; PI, propidiumiodide.

 

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© The Company of Biologists Ltd 2006