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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01214

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Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

¹ Developmental Biology Program, The Saban Research Institute of Childrens' Hospital Los Angeles, Departments of Pathology and Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
² Cardiovascular Division, Department of Medicine and the Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA

View larger version (126K): [in a new window]   Fig. 1. Expression of Alk2 and its putative ligands during cardiac development at E11.5. Radioactive in situ hybridization analysis of transverse sections through the cardiac OFT. Alk2 mRNA (A) displays a very similar expression pattern to Plxna2 (C) and Ednra (D), indicating expression by cardiac neural crest cells (Brown et al., 2001; Charite et al., 2001). Arrows indicate the positive hybridization signal (A,C,D). For comparison, cardiac neural crest cells were fate mapped using the WNT1-Cre/R26R reporter assay (Jiang et al., 2000) (B). An arrow indicates NCCs in the OFT region (blue signal). Bmp4 (F), Bmp5 (G), Bmp6 (H), Bmp7 (I) and Tgfb3 (K) are expressed in the myocardium, while Bmp2 (E) and Tgfb2 (J) are also expressed in the mesenchyme of OFT cushions. Scale bar: 200 µm.

View larger version (63K): [in a new window]   Fig. 2. Cardiac OFT defects in newborn Alk2/Wnt1-Cre mice. (A) Functional domains of the ALK2 protein and schematic presentation of the Alk2 locus, the floxed and knockout alleles. Cre-mediated recombination results in excision of exon 7 flanked by the loxP sites (Alk2Flox allele; red arrowheads), which encodes the Smad interacting domain (the L45 loop) and a crucial part of the kinase domain. This recombination results in generation of a null allele (Alk2KO), which is biochemically inactive (Dudas et al., 2004). Blue arrows depict primers used to identify the Alk2Flox allele. Intron 6 and intron 7 specific primers (green and blue arrows, respectively) were used to identify the Alk2KO allele. (B) Homozygous floxed Alk2 (Alk2Flox/Flox) mice were bred with Wnt1-Cre mice that are heterozygous for the Alk2 knockout allele (Alk2KO) to produce a NC-specific deletion of exon 7. (C) Normal anatomy is seen in the control heart with the aorta (Ao) arising to the left and dorsal to the main pulmonary artery (PA) (left). The Alk2 mutant displays an abnormal single OFT, persistent truncus arteriosus (TA) (right). Moreover, the right ventricle (RV) of the mutant heart (right) is significantly larger than that of the control (left), and the brachiocephalic artery (BC) is short in the Alk2 mutant (C, frontal view and D, lateral view). Atria have been removed to facilitate better visualization of the OFTs. LV, left ventricle; RV, right ventricle. RS, right subclavian artery; RC, right common carotid artery; LC, left subclavian artery. (E) Hematoxylin and Eosin staining shows that the thymus is unaffected in Alk2/Wnt1-Cre mutants (right) compared with controls at E14.5 (left). Scale bar: 100 µm.

View larger version (121K): [in a new window]   Fig. 3. Abrogation of Alk2 in neural crest cells leads to persistent truncus arteriosus. Hematoxylin and Eosin-stained sections of control (A-F) and Alk2 mutant (G-L) littermate embryos at E14. In controls (A-F), pulmonary artery (PA) and the ascending aorta (Ao) are separated by the conotruncal (aortico-pulmonary) septum, and the right and left ventricles by the interventricular septum (VS). In Alk2 mutants (G-L), the conotruncal septum fails to form, i.e. they display persistent truncus arteriosus (TA; G,H), which is always associated with a ventricular septal defect (VSD, K). Both the atrioventricular and semilunar valves appear normal. An approximate plane of the shown sections is indicated on the schematic drawings (left). Scale bar: 200 µm.

View larger version (104K): [in a new window]   Fig. 4. Abnormal pharyngeal arch arteries in Alk2/Wnt1-Cre mutants. Left lateral view after intracardiac ink injection to visualize developing pharyngeal arch arteries at E10.5 (A, B) and at E11.5 (C,D) in controls (A,C) and Alk2 mutants (B,D). The pharyngeal arch arteries are numbered. Note regression of arteries 3 and 6 in the Alk2 mutant at E11.5. Immunohistochemistry for the smooth muscle cell marker, -smooth muscle actin at E11.0 in control (E,G) and Alk2 mutant samples (F,I). The high-power pictures show the 6th pharyngeal arch artery in the control (G,H) and mutant (I,J). There is diffuse discontinuous brown staining around the 3rd and 6th pharyngeal arch arteries at E11.0 in mutants (I, arrows in J) when compared with controls (G,H). Neural crest cells were fate mapped using the WNT1-Cre/R26R reporter assay (H,J; green) to show that in controls (H) and in mutants (J) the aortic arch arteries are surrounded by NCCs. Scale bars: 200 µm in E,F; 50 µm in G-J.

View larger version (96K): [in a new window]   Fig. 5. Early migration of NCCs in Alk2/Wnt1-Cre mutants. Expression of NCC markers Msx1 (A,B), Foxd3 (C,D) and Sox10 (E,F) in controls (A,C,E) and Alk2 mutants (B,D,F) at E9.0. The expression pattern of Msx1 (A,B; arrows; dorsal view) in the dorsal neural tube is indistinguishable between controls (A) and Alk2 mutants (B). Both Foxd3 (C,D; dorsal view) and Sox10 (E,F; lateral view) are expressed in all three trajectories of migrating NCCs (arrows in C-F) and show no differences between the Alk2 mutants and controls.

View larger version (75K): [in a new window]   Fig. 6. Fate mapping of cardiac NCCs in Alk2/Wnt1-Cre mutants. Embryos carrying the R26 Cre reporter were stained for the ß-galactosidase activity at E10.5 (A-C) and at E11.5 (D-F) to identify cells derived from the NC (A,D, controls; B,C and E,F, Alk2 mutants). Arrows indicate the OFT. In the control, blue staining is more proximal than in the mutant. Frontal sections through the conotruncal region of control (G-I) and Alk2 mutant (J-L) at E11.5. Level and orientation of each section has been indicated in D and E. In control sections, arrows indicate positively staining NC-derived cells that contribute to formation of conotruncal cushions. In mutants, arrows highlight near-complete absence of NC-derived cells in the more proximal region (level a) and significant reduction of positively staining cells in more distal regions (levels b and c). M-O, the whole-mount staining of the control (M) and Alk2 mutant (N,O) hearts at E13. Both the aorta and pulmonary trunk stain positive for cells derived from NCCs (blue staining) in the control (M). In Alk2 mutants, the persistent truncus arteriosus shows positively staining cells, albeit with variable intensity (N,O). Scale bars: 200 µm in G-L.

View larger version (97K): [in a new window]   Fig. 7. Cell proliferation and marker gene expression in the cardiac OFT. Cell proliferation was analyzed in transverse sections of controls (A) and Alk2/Wnt1-Cre mutants (B) by using BrdU incorporation assay. The mesenchyme of OFT cushions (arrows) is significantly reduced in size in Alk2 mutants (B) when compared with the corresponding tissues in controls (A). (A',B') High-magnification views of the OFT cushions indicated with arrows in A and B. Comparison of the proliferation indices for OFT cushion cells indicated that cell proliferation did not significantly differ between these two genotypes (n=3). In Alk2/Wnt1-Cre mutants, the level of Plxna2 (arrows, marker for NCCs) expression is diminished at E11.0 (D) compared with controls (C). Msx1, an effector of BMP signaling, is expressed in the proximal OFT mesenchyme (arrows) both in controls (E) and ALK/WNT1-Cre mutants (F). However, only the control samples display Msx1 expression in the distal OFT mesenchyme (arrowhead in E). Scale bars: in A, 200 µm for A,B; in A', 100 µm for A',B'; in C, 200 µm for C-F.