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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01287

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Foxp1 regulates cardiac outflow tract, endocardial cushion morphogenesis and myocyte proliferation and maturation

Bin Wang^1,*, Joel Weidenfeld^2,*, Min Min Lu^2,, Shanna Maika¹, William A. Kuziel¹, Edward E. Morrisey^2,3,, and Philip W. Tucker^1,,

¹ Department of Molecular Genetics and The Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
² Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
³ Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (49K): [in a new window]   Fig. 1. Inactivation of Foxp1 in mice. (A) Schematic of Foxp1-targeting construct showing probe and predicted PstI digest pattern. (B) Southern blot analysis of yolk sac DNA using the probe indicated in A. (C) Immunohistochemistry of wild-type (WT) and Foxp1 null (–/–) hearts at E13.5 using previously characterized Foxp1 antibody (Lu et al., 2002). Note lack of Foxp1 expression in –/– heart tissue. (D) Distribution table of embryonic lethality and lack of postnatal survival in Foxp1 homozygous null embryos and mice. (E) Wild-type and Foxp1–/– embryos at E14.5, showing the perivascular hemorrhage and edema in –/– embryos. Scale bars: 50 µm.

View larger version (105K): [in a new window]   Fig. 2. Histology of Foxp1–/– hearts at E11.5 and E14.5. H+E staining was performed on wild-type (A,C,E,G) and Foxp1–/– (B,D,F,H) embryos to examine cardiac morphology at E11.5 (A-D) and E14.5 (E-H). Foxp1–/– hearts have a thin myocardial compact zone (compare C and D,G,H, brackets). Foxp1–/– hearts also show ventricular septation defects (vsd) (F). Scale bars: 400 µm in A,B; 200 µm in C,D,G,H; 800 µm in E,F.

View larger version (92K): [in a new window]   Fig. 3. Expression of Foxp1 protein during cardiac development. A Foxp1-specific antibody (Lu et al., 2002) was used to determine Foxp1 protein expression at E9.5 (A,B), E11.5 (C,D), E12.5 (E,F,I,J), and E14.5 (G,H,K,L) in the heart and outflow tract vessels. Foxp1 expression is observed in the ventral aspect of the foregut endoderm and the myocardium (A,B, arrows). Foxp1 expression is observed throughout the heart, including myocardium, endocardium and endocardial cushion tissue at E11.5 (C,D). Foxp1 is expressed in cells underlying the mesenchyme of the forming endocardial cushion (C, arrows). Foxp1 is expressed in a decreasing gradient from the compact zone to the trabecular zone as early as E11.5 through E14.5 (D-F,H; note arrows in E,F and H showing decreased expression in trabecular myocardium). By E14.5, expression in endocardial cushion mesenchyme is extinguished but remains high in the overlying endocardial cells (G, arrow). Foxp1 expression is observed in the endothelium of the aorta and pulmonary artery at E12.5 (I) and E14.5 (K). Foxp1 expression is also observed in the VSMCs of these vessels at E12.5 and E14.5 (J,L). The wall of the ductus arteriosus can be observed in J,L. FE, foregut endoderm; myo, myocardium; endo, endocardium; Ao, aorta; Pa, pulmonary artery. Scale bars: 200 µm in A,G,H; 100 µm in B,C-E,I-K; 50 µm in F, 150 µm in L.

View larger version (104K): [in a new window]   Fig. 4. Foxp1–/– embryos have severe defects in outflow tract development. H+E staining of embryonic sections of wild-type (A-C,G-I) and Foxp1–/– (D-F,J-L) were performed to characterize defects in outflow tract development. Foxp1 null embryos show PTA at E11.5 (E, arrow) and at E14.5 (F, arrow). Of Foxp1 null embryos, 20% have DORV, with both aorta and pulmonary artery arising from the right ventricle (J-L), whereas wild-type littermates exhibit the correct septation of these vessels, with the aorta arising from the left ventricle and the pulmonary artery arising from the right ventricle (G-I). RV, right ventricle; LV, left ventricle; AO, aorta; PA, pulmonary artery. Scale bars: 400 µm in A-F; 600 µm in G-L.

View larger version (68K): [in a new window]   Fig. 5. Defects in endocardial cushion and valve formation in Foxp1–/– hearts. H+E staining of embryonic sections of wild-type (A,C) and Foxp1–/– (B,D) were performed to characterize defects in endocardial cushion and valve development at E14.5. By E14.5, endocardial cushion mesenchyme in wild-type heart valves has already begun to regress to form the mature pulmonary (A) and atrial-ventricular heart valves (C). In Foxp1–/– hearts, the cushion mesenchyme has not regressed as far and the cushions appear as large bulges (B,D, arrows). At E11.5, TUNEL assays reveal decreased levels of apoptosis in the outflow tract endocardial cushion mesenchyme in Foxp1–/– hearts (E) relative to wild type (F). At E11.5, cell proliferation as determined by phospho-histone H3 immunostaining is unchanged in Foxp1 outflow tract cushions (G,H). Nfatc1 expression (J,L, arrowheads) in Foxp1–/– hearts persists in the endocardial cushion mesenchyme in both pulmonary (J) and mitral cushions (L) compared with wild-type pulmonary (I) and mitral (K) cushions at E14.5. Quantification of both TUNEL-positive cells (M) and phosphohistone H3-positive cells (N) for data shown in E-H. E,G and F,H are adjacent histological sections. Scale bars: 400 µm in A-D; 200 µm in E-L.

View larger version (60K): [in a new window]   Fig. 6. Sox4 is downregulated in Foxp1 null hearts. In-situ hybridization for Sox4 (A-E), Foxc1 (H,I) and Foxc2 (J,K) and immunohistochemistry for fibronectin (F,G) was performed on wild-type (A,D,F,H,J) and Foxp1–/– (B,C,E,G,I,K) E11.5 (A-C) and E14.5 (D-K) embryo sections. Sox4 gene expression is significantly decreased in the outflow tract region of Foxp1–/– hearts at both E11.5 and E14.5 (A-C), while Foxc1, Foxc2 and fibronectin expression is unchanged. Scale bars: 800 µm in D,E,H-K; 400 µm in A-C,F,G.

View larger version (107K): [in a new window]   Fig. 7. Cell proliferation and expression of cyclin-dependent kinase inhibitors in Foxp1–/– hearts. Immunohistochemistry was performed on wild-type (A,C,E,G) and Foxp1–/– (B,D,F,H) hearts at E13.5 using antibodies for phospho-histone H3 (A,B), p21 (C,D), p27 (E,F), and p57 (G,H). Significant increases in phospho-histone H3 (B, red arrowheads) and p21 staining (D, red arrowheads) were observed, while a decrease in p27 expression was observed in the compact zone of Foxp1–/– hearts (F, red arrow). Quantifying the number of phospho-histone H3 positive cells in the trabecular zone for wild-type and Foxp1–/– hearts shows an approximately threefold increase in the number of mitotic cells in the trabecular zone of Foxp1–/– hearts but no change in the compact zone (I). Identical analysis of p21 expression levels reveals an approximate fourfold increase in trabecular myocardium but no significant difference in compact myocardium (J). p27 levels were decreased by almost 50% in Foxp1–/– compact myocardium, while levels in trabecular myocardium were unchanged (K). Scale bar: 200 µm.

View larger version (90K): [in a new window]   Fig. 8. Myocardial differentiation is disrupted in Foxp1–/– hearts. In-situ hybridization was performed using probes specific for Irx3 (A,B), Anf (C,D) and N-myc (E-H) on wild-type (A,C,E,G) and Foxp1–/– (B,D,F,H) hearts at E11.5 (E,F) and E14.5 (A-D,G,H). Irx3 expression is expanded to encompass almost all compact and trabecular myocardium (B). N-myc expression was significantly reduced in the ventricular wall of Foxp1–/– embryos (compare E,F,G,H). H+E staining of wild-type and Foxp1–/– hearts at E11.5 shows a thin compact zone in the myocardial wall of the ventricle wall of Foxp1–/– hearts (I,J, brackets). Transmission electron microscopy was performed on E11.5 wild-type (K) and Foxp1–/– (L) hearts. Wild-type compact zone myocardium shows a laminated appearance, while Foxp1–/– heart compact zone myocardium has a disorganized appearance (compare bracketed regions). Scale bars: 250 µm in A,B; 400 µm in C-F; 800 µm in G,H; 100 µm in I,J.