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First published online 19 April 2006
doi: 10.1242/dev.02374

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Distinct cardiac malformations caused by absence of connexin 43 in the neural crest and in the non-crest neural tube

¹ Leon H. Charney Division of Cardiology, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.
² Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
³ Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.

View larger version (25K): [in a new window]   Fig. 1. Strategy for tissue-restricted conditional knockout (CKO) of Cx43. (A) Schematic representation of wild-type, floxed and floxed-out alleles, demonstrating the locations of NcoI sites, the probe used for Southern blotting (solid bar) and primer locations for PCR (small arrows). ORF, open reading frame; UTR, untranslated region. (B,C) Mating strategies for the generation of Wnt1-(B) and P3pro-Cre-(C) mediated CKO mice. Ctrl, control; flox, floxed; fo, floxed-out (Cx43-null). (D) Southern blotting of genomic DNA samples from tails of offspring from P3proCre+:Cx43fo/wt x P3proCre-:Cx43flox/wt matings. NcoI-digested genomic DNA yields a 6.5 kb wild-type band, a 5.4 kb floxed band and a 4.3 kb floxed-out band. Samples shown are from P3proCre-:Cx43flox/fo (lane 1), P3proCre+:Cx43fo/wt (lane 2), P3proCre+:Cx43flox/fo (Cx43-PCKO; lane 3) and P3proCre-:Cx43flox/wt pups (lane 4). (E) PCR samples showing similar genotypes to those in corresponding lanes in D. One primer pair is located entirely within exon 2 and generates a 220 bp PCR band from wild-type DNA and a 180 bp band from the floxed allele. A second primer pair generates a 550 bp product from the floxed-out allele and no product from either wild-type or floxed alleles.

View larger version (81K): [in a new window]   Fig. 2. Neural crest-specific knockout of Cx43 in Cx43-WCKO embryos. (A) Low-power, sagittal section of a wild-type E9.5 embryo demonstrating first, second and third pharyngeal arches. (B,C) Immunostaining for Cx43 (green stain) in high-power images of the first pharyngeal arch (PA1) (B) and the third pharyngeal arch (PA3) (C), in the E9.5 wild-type embryo. (D) Low-power image of an E9.5 Cx43-WCKO embryo. (E,F) High-power images of PA1 (E) and PA3 (F) in the E9.5 Cx43-WCKO embryo immunostained for Cx43. Cx43 signal is nearly absent in PA1 and PA3 in the Cx43-WCKO embryos in contrast to wild-type littermates. (G,H) Heart sections from E10.5 wild-type (G) and Cx43-WCKO (H) embryos immunostained for Cx43. Expression of Cx43 in the Cx43-WCKO heart is preserved at this stage. Propidium iodide nuclear staining is in red. Scale bar: 320 µm for A,D; 20 µm for B,C,E,F; 40 µm for G,H.

View larger version (71K): [in a new window]   Fig. 3. Coronary anomalies in Cx43-WCKO neonatal hearts. (A-D) A typical pattern of coronary deployment in control mouse hearts is characterized by single left (A) and right (C) coronary ostia, each giving rise to arteries that bifurcate into left (B) and right (D) myocardial and septal branches. (E-H) Coronary abnormalities in Cx43-WCKOs included separate right septal (E) and myocardial branch ostia (F) in one heart, an accessory coronary (AC) originating from the non-coronary sinus in another heart (G), and tunneling of the left coronary artery through the wall of the aorta (arrows, H) in a third heart. Ao, aorta; PA, pulmonary artery; LC, left coronary artery; S, septal branch; M, myocardial branch; RC, right coronary artery; RV, right ventricle. Scale bar: 100 µm.

View larger version (93K): [in a new window]   Fig. 4. Right ventricular outflow tract (RVOT) morphology of Cx43-WCKO hearts is grossly normal at birth. (A-C) Whole-mount images of control (A), Cx43-WCKO (B) and Cx43fo/fo (germline Cx43-null) (C) neonatal hearts. (D-F) Hematoxylin and Eosin-stained sections from control (D), Cx43-WCKO (E) and Cx43fo/fo (F) neonatal hearts. The Cx43-WCKO RVOT (arrow) appears similar to that of its littermate controls and lacks the infundibular bulging and exaggerated trabeculation (asterisk) seen in germline Cx43 KO hearts. P, pulmonary trunk; RVOT, right ventricular outflow tract. Scale bar: 1 mm.

View larger version (61K): [in a new window]   Fig. 5. Loss of Cx43 expression in Cx43-PCKO embryos involves the cardiac neural crest distribution. (A) Low-power image of a wild-type E9.5 embryo in cross-section demonstrating first, second and third pharyngeal arches. (B,C) Corresponding high-power images of the first pharyngeal arch (PA1) (B) and the third pharyngeal arch (PA3) (C) in the E9.5 wild-type embryo immunostained for Cx43 (green stain). (D) Low-power image of an E9.5 Cx43-PCKO embryo. (E,F) Corresponding high-power images of PA1 (E) and PA3 (F) in the E9.5 Cx43-PCKO embryo immunostained for Cx43. Cx43 signal is similar in PA1, but reduced in PA3 at E9.5 in the Cx43-PCKO embryos compared with wild-type littermates. (G,H) Heart sections from E10.5 wild-type (G) and Cx43-PCKO (H) embryos immunostained for Cx43. Expression of Cx43 in the Cx43-PCKO heart is preserved at this stage. Propidium iodide nuclear staining is in red. Scale bar: 200 µm for A,D; 20 µm for B,C,E-H.

View larger version (80K): [in a new window]   Fig. 6. Right ventricular morphology in Cx43-PCKO hearts is grossly abnormal. (A) Grossly visible bulges flanking the OFT are seen in Cx43-PCKO hearts (black arrows) and occasionally focal bulging segments at the right ventricular apex (arrowhead). (B) Detailed view of the RVOT in a Cx43-PCKO heart demonstrates grossly bulging infundibular contour. (C) Although most Cx43-PCKO hearts have infundibular bulges (arrows), many (including this heart) lack the bulging apical segment seen in A. (D) The RVOT region of the heart in C is markedly distorted. (E,F) Control and (G,H) Cx43fo/wt (heterozygous Cx43-null) hearts have grossly normal right ventricular and OFT morphology. Infundibular contours (outlined) form a triangular shape in the control and Cx43fo/wt hearts as they course towards the pulmonary trunk. (I,J) Infundibular bulges (arrows) in the Cx43-PCKO OFT are similar to those of the Cx43fo/fo (germline Cx43-null) heart. (K) Oblique view of the Cx43-PCKO RVOT demonstrates infundibular bulging above the level of the pulmonic valve. (L) By contrast, the contour of the wild-type RVOT tapers as it approaches the pulmonary trunk. P, pulmonary trunk; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract. Scale bar: 1.2 mm for A,E,G,I,L; 750 µm for C,K; 400 µm for B,D,F,H,J.

View larger version (126K): [in a new window]   Fig. 7. Infundibular abnormalities are first seen at E15.5 in Cx43-PCKO hearts. (A-C) Hematoxylin and Eosin stained sections from E15.5 control (A), Cx43-PCKO (B) and Cx43fo/fo (C) hearts. Infundibular bulging is first apparent in the Cx43-PCKO and germline KO hearts at this stage (arrows in B and C). (D-F) Sections from control (D), Cx43-PCKO (E) and Cx43fo/fo (F) E17.5 hearts. The RVOT of the Cx43-PCKO heart is markedly deformed, as is that of the Cx43fo/fo heart (arrows in E and F, respectively). (G-I) Sections from control (G), Cx43-PCKO (H) and Cx43fo/fo (I) E17.5 hearts at the level of the LVOT. The Cx43-PCKO LVOT, like that of the Cx43fo/fo heart, appears similar to controls, although the RV-free walls of both the Cx43-PCKO and Cx43fo/fo hearts are thinned in comparison with controls (asterisks in H and I, respectively). (J-L) Sections through the RVOT of neonatal control (J), Cx43-PCKO (K) and Cx43fo/fo (L) hearts. The Cx43-PCKO RVOT is grossly dilated, heavily trabeculated (arrows) and bulges above the level of the pulmonic valve (arrowhead), unlike the control RVOT but similar to that of Cx43fo/fo hearts. Neo, neonatal. Scale bar: 450 µm for A-C,N; 700 µm for D-M,O.

View larger version (100K): [in a new window]   Fig. 8. Coronary patterning is altered in Cx43-PCKO hearts. (A) One neonatal Cx43-PCKO heart has a right septal branch (RS) that originates from the left coronary artery. (B-E) Two separate coronary ostia are seen in the left coronary sinus of one of the Cx43-PCKO neonatal hearts (B,C). One ostium (LCO1) gives rise to a septal artery (B); the other (LCO2) gives rise to a large, aneurysmal artery that traverses the bulging tissue of the infundibulum (C,D). This mutant heart also has a blistering appearance of the right ventricular lateral wall, possibly consistent with subepicardial coronary plexuses described in the germline Cx43-null heart (arrows, E) (Walker et al., 2005). (F-H) Another neonatal Cx43-PCKO heart has dual right coronary ostia. One ostium (RCO1) leads to a large septal artery (F). The second right coronary ostium (RCO2) gives rise to a septal branch and a myocardial tributary (G), which subsequently divides into branches that cross through the RV infundibulum (arrows, H). (I-K) Tunneling of the right coronary artery (arrows) through the wall of the aorta is shown in an E17.5 Cx43-PCKO heart. (L) Two out of five neonatal heterozygous Cx43-null (Cx43fo/wt) hearts demonstrated coronary artery branching at the ostium. Ao, aorta; LC, left coronary artery; LCO, left coronary ostium; LM, left myocardial branch; LS, left septal branch; LV, left ventricle; M, myocardial branch; PA, pulmonary artery; RA, right atrium; RC, right coronary artery; RCO, right coronary ostium; RV, right ventricle; S, septal branch. Scale bar: 225 µm for A-D; 112.5 µm for E,G-L; 56 µm for F.

View larger version (143K): [in a new window]   Fig. 9. Exuberant delamination of neuroepithelial cells in E11.5 Cx43-PCKO embryos. (A-F) P3pro-Cre-expressing control (A) and Cx43-PCKO E11.5 embryos (D), crossed into the EYFP Cre reporter line demonstrate extensive Cre activity throughout the neural tube (nt) and dorsal root ganglia (drg). High-power views of the dorsal (B) and lateral (C) aspects of the control neural tube shown in A demonstrate a sharp boundary at the neuroepithelial border (arrowheads). No cells crossing this boundary are seen. In contrast to controls, neural tube cells in the E11.5 Cx43-PCKO embryo are delaminating from the dorsal (E) and lateral (F) aspects of the neural tube (arrows; asterisked arrow indicates an actively delaminating cell). (G-I) As observed in the Cx43-PCKOs, delamination of cells from Cx43 germline KO (Cx43fo/fo) neural tubes (G) is detected along the dorsal (H) and lateral (I) surfaces. (J-O) Wnt1-Cre activity is limited primarily to the dorsal regions of E11.5 EYFP-expressing control (J) and Cx43-WCKO (M) neural tubes. Labeled cells are not seen outside either dorsal or lateral aspects of the Wnt1-Cre+ control (K,L) or Cx43-WCKO (N,O) neural tubes. EYFP fluorescence appears green, propidium iodide to highlight cell nuclei is red and their overlap results in yellow signal. Scale bar: 200 µm for A,D,G,J,M; 50 µm for all other panels.

View larger version (118K): [in a new window]   Fig. 10. Increased abundance of neuroepithelium-derived cells in the E11.5 Cx43-PCKO embryo. (A) Wild-type E11.5 expression pattern of P3pro-Cre+:Cx43+/+, EYFP-labeled cells (green signal) are limited to the neural tube, dorsal root ganglia (drg), peripheral nerves (pn), sympathetic ganglia (sg) and small circumscribed collections of labeled cells flanking the trachea (t) and esophagus. Sections in A-F are counterstained with propidium iodide to highlight the nuclei; overlap with EYFP appears as yellow signal. (B) Labeled cells flanking the trachea are shown migrating into the OFT of a control E11.5 embryo. (C) Section from an E11.5 Cx43-PCKO EYFP embryo at the level of the heart, showing an increased abundance of labeled cells outside of the neural tube in comparison with the control littermate. (D) Labeled cells are abundant in and around the tracheal region adjacent to the OFT in the Cx43-PCKO embryo at E11.5. (E,F) Labeled cells in sections from E11.5 Wnt1-Cre+ control (E) and Cx43-WCKO embryos (F) at the level of the heart share a similar distribution to that of P3pro-Cre+ controls, except in the neural tube where the Wnt1-Cre+ cells have a more limited distribution. (G) Radioactive in situ hybridization of E11.5 control embryos shows plexin A2 mRNA (pink) distributed in the neural tube (arrowhead) and in the OFT (arrow), as well as in paratracheal tissue adjacent to the OFT. (H) In contrast to the control embryos, Cx43-PCKO embryos express higher levels of plexin A2 (pink) in the neural tube (arrowhead), OFT (arrow) and paratracheal tissue, as well as elsewhere in the embryo. (I,J) Immunostaining for the presence of Pax3 (red) in control (I) and Cx43-PCKO (J) sections (indicated by arrows) at E11.5 reveals an unchanged expression pattern in the mutant embryos. (K) Cre recombinase expression (green) is absent in control embryos. (L) Cre expression is seen primarily at the dorsal aspect of the neural tube in Cx43-PCKO embryos at this stage (arrows). Arrowheads indicate the ventral boundary of the neural tube. Scale bar: 200 µm for A-F; 100 µm for G,H; 400 µm for I-L.

View larger version (103K): [in a new window]   Fig. 11. Extensive infiltration of labeled cells into the E15.5 Cx43-PCKO heart. (A) Short axis section through the RV infundibulum in an E15.5 P3pro-Cre+ control embryo showing labeled cells primarily limited to the area adjacent to the aortic valve. (B) Higher magnification of the infundibular myocardium in A (boxed) demonstrates only rare labeled cells. (C) RVOT of an E15.5 Cx43-PCKO embryo showing abundant labeled cells infiltrating the myocardium. (D) Higher magnification of the indicated region in C demonstrates numerous labeled cells in the myocardial wall and trabeculae. (E-H) Short axis sections through the RV infundibulum in a Wnt1-Cre+ control (E,F) and a Cx43-WCKO heart (G,H) reveal a limited distribution of labeled cells (arrows) similar to that seen in the P3pro-Cre+ control. F,H are higher magnifications of the boxed areas in E,G. Ao, aorta; RV, right ventricle. Scale bar: 400 µm for A,C,E,G; 50 µm for B,D,F,H.

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