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Fig. 1. Position of the putative cardiac neural crest and experimental design to analyze its contribution to the cardiovascular system. (A) Diagram of a neurula stage Xenopus embryo (stage 17) viewed from the dorsal side (anterior to top), with outline of the segments of the neural crest (NC) ablated along the anteroposterior axis (1-3). (B-D) Stage 17 embryos with the region of the three ablated NC domains indicated. (E-H) In situ hybridization for the NC marker Sox10 in control and NC-ablated embryos at stage 25. (E) In control embryos, Sox10 is expressed within the four streams of the migrating cranial NC: the mandibular (a), hyoid (b), anterior branchial (c) and posterior branchial (d) NC. (F) After ablation of NC domain ‘#1’, the most anterior NC stream (a) is lost. (G) Ablation of NC domain ‘#2’ results in loss of the three most posterior NC streams, without affecting the more anterior mandibular NC stream (a). (H) After ablation of NC domain ‘#3’, all four streams of the migrating cranial NC form normally. (I-N) Position of the ablated/transplanted NC domain at stage 17 with respect to adjacent tissues. The presumptive otic placode (I-K) as revealed by Sox9 expression (arrows) is unaffected after NC ablation. The ablated NC domain spans a region extending from the first to the fourth somite (L-N) as revealed by MyoD expression (brackets). (O) Experimental design to analyze NC contribution to the cardiovascular system. Two-cell stage embryos were injected in the animal pole with mRNA encoding RFP. At stage 17, the RFP-labeled NC is transplanted onto the equivalent region of an unlabeled host embryo (NC graft). (P,Q) RFP-labeled NC graft at stage 17. (R-U) At stage 25, cells derived from the RFP-labeled NC graft are confined to the three most posterior streams of cranial NC (b, c and d), and completely overlap with Sox10 expression as seen in the whole embryo (R,T) and in sections (S,U). The line in R and T indicates the level of the sections shown in S and U, respectively. Scale bars: 100 μm.
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Fig. 2. RFP-labeled NC contributes to the aortic sac and arch arteries and is dispensable for outflow tract septation. (A-D) Cells derived from a stage 17 RFP-labeled NC graft populate the wall of the aortic arch arteries at stage 45 and never populate the outflow tract (OFT). B-D are a higher magnification of the boxed area in A. Ventral views, anterior to top. (E-H) Four views of the same Xenopus embryo showing that the RFP-positive cells form a very sharp boundary (arrowheads) and do not overlap with the myocardium muscle cells, as labeled with MF20 antibody. (I-L) At stage 48, RFP-labeled NC-derived cells are confined to the large arteries (LCC, left carotid canal; LPC, left pulmocutaneous canal; RCC, right carotid canal; RPC, right pulmocutaneous canal) as seen in the whole embryo (J) and in sections (K,L). (M-X) Transverse sections of stage 41 embryos showing Nkx2.5 and Sox8 expression in control and manipulated embryos. (M-N) In control embryos, Nkx2.5 is expressed throughout the myocardium [outflow tract and ventricle (V)], whereas Sox8 is confined to the spiral septum (arrows). Sox8 is also detected in the NC-derived head mesenchyme (asterisks). (O-P) NC ablation does not prevent outflow tract septation as revealed by Sox8 expression (arrows). These embryos lack the aortic sac, resulting in a dorsal expansion of Nkx2.5. (Q-T) The RFP-labeled NC-derived cells are detected in the aortic sac and arch arteries (arrowheads) but not in the spiral septum of the outflow tract (arrows). U-X are higher magnification views of Q-T. Scale bars: 100 μm.
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Fig. 3. Molecular characterization of the primary and second heart fields. (A-U) Developmental expression of Nkx2.5, Tbx5 and Tbx20 at stages 25, 30, 33 and 35 as indicated. The horizontal lines indicate the predicted position of primary heart field (PHF) and second heart field (SHF). All three genes are detected in the PHF, whereas the SHF expresses both Nkx2.5 and Tbx20 but is negative for Tbx5 expression. At later stages, the developing outflow tract (OFT), a derivative of the SHF, remains Tbx5 negative. (A,C,E,G,I,K,M-U) Lateral views, anterior to right. (B,D,F,H,J,L) Ventral views, anterior to top. V, ventricle. Scale bars: 100 μm.
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Fig. 4. The PHF contributes to the ventricle. (A) Experimental design to analyze the relative contribution of the PHF and SHF to the developing heart. Four-cell stage Xenopus embryos were injected in the dorsal marginal zone (DMZ) with mRNA encoding GFP. At stage 25, the GFP-labeled PHF or SHF was transplanted onto the equivalent region of an unlabeled host embryo (PHF graft or SHF graft). (B-D) Ventral view (anterior to top) of a host embryo at stage 26 shows the position of GFP-labeled PHF graft. (E-G) At stage 41, the GFP-labeled PHF-derived cells are detected in the developing heart. Ventral view, anterior to top. (H-J) In a transverse section, the GFP-labeled PHF-derived cells are largely confined to the ventricle. OFT, outflow tract; V, ventricle. Scale bars: 100 μm.
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Fig. 5. The SHF contributes to the outflow tract and forms the spiral septum. (A-C) Xenopus embryos at stage 25 immediately after ablation of the SHF and hybridized with Nkx2.5, Tbx5 and Tbx20 probes indicate that the ablated SHF is anterior to the Tbx5 expression domain. (D-F) Expression of Nkx2.5, Tbx5 and Tbx20 in stage 30 control embryos. (G-I) After ablation of the SHF, embryos show an overall reduction in Nkx2.5 expression and a loss of the most anterior cardiac domain of Tbx20 (arrow). Tbx20 is also expressed in the cement gland (arrowhead). Tbx5 expression is largely unaffected. However, SHF-ablated embryos show a delay in the fusion of the PHF at the midline. (J-O) SHF graft restores the anterior domain of Tbx20 expression at stage 30 (arrow, L), and the fusion at the midline of the PHF (K). (M-O) Brightfield views showing the position of the GFP-labeled SHF graft. (P,Q) Fluorescence and brightfield views of a stage 41 embryo after SHF graft at stage 25 showing GFP-positive cells in the developing heart. (A-Q) Ventral views, anterior to top. (R-Y) Transverse sections of similar stage 41 embryos. (R,S) Expression of Nkx2.5 and Tbx5 in stage 41 control embryos. Whereas Nkx2.5 is expressed throughout the myocardium (outflow tract and ventricle), Tbx5 is restricted to the ventricle, establishing a sharp boundary with the outflow tract (arrowheads). (T,U) After SHF ablation, the overall size of the cardiac tissue is extremely reduced but it expresses both Nkx2.5 and Tbx5, suggesting that it is likely to be PHF derived. (V-Y) GFP-labeled SHF-derived cells are detected in the wall of the outflow tract (arrowheads), part of the ventricle and in the spiral septum (arrows). Labeling of the ventricle suggests that the segregation of the PHF and SHF is likely to be incomplete at the time of transplantation (Gessert and Kühl, 2009). In these embryos, the pharyngeal endoderm is also GFP labeled (asterisks) because the transplanted SHF graft contains both mesoderm and endoderm layers (see Table 1). OFT, outflow tract. Scale bars: 100 μm.
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Fig. 6. The NC and SHF contribute to distinct, non-overlapping lineages of the cardiovascular system. (A-X) RFP-labeled NC and GFP-labeled SHF were sequentially transplanted into the same unlabeled host Xenopus embryo at stage 17 and stage 25, respectively. The progeny of the labeled grafts were analyzed at different time points, in the whole embryo at stage 27 (A-H) and stage 35 (I-P) and on sections at stage 41 (Q-X). (A-D,I-L) Lateral views, anterior to right. (E-H,M-P) Ventral views, anterior to top. Sox8 expression at stage 41 is detected in the spiral septum (black arrow), a tissue derived from the GFP-labeled SHF (white arrows). GFP-labeled SHF also contributes to the wall of the outflow tract and to the proximal part of the ventricle. RFP-labeled NC-derived cells are detected in the aortic arch arteries (arrowheads) and in the NC-derived head mesenchyme. Red blood cells in the ventricle show non-specific autofluorescence (asterisks). U-X are higher magnification views of Q-T. Scale bars: 100 μm.
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Fig. 7. The relative contribution of the cardiac NC and SHF to the Xenopus cardiovascular system. (A) Hematoxylin and Eosin (H/E) staining of a transverse section through the heart of a stage 50 embryo. AA, aortic arch arteries; AS, aortic sac; LA, left atrium; OFT, outflow tract; RA, right atrium; SS, spiral septum; V, ventricle. Scale bar: 100 μm. (B) An adjacent section stained with MF20 antibody highlights the very sharp boundary between the muscle cells in the myocardium of the outflow tract and the aortic sac. (C) The Xenopus developing heart. (D) The muscle marker MF20 is expressed in muscle cells of the myocardium, including outflow tract and ventricle, but is excluded from the spiral septum. (E) The structures derived from the cardiac NC (CNC, red), the SHF (yellow) and the PHF (blue) are indicated based on our findings. PHF and SHF overlapping regions that result from the incomplete segregation of the two heart fields at the time of transplantation are in green.
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Fig. S1. DiI fate mapping of the cardiac NC at stage 17/ (A,B) At stage 27, DiI-labeled NC cells are populating the branchial arches (arrows). Lateral view, dorsal to top, anterior to right. (C,D) Ventral view of a stage 45 embryo showing the restriction of the DiI-positive cardiac NC cells to the aortic arch artery on the right side. (E,F) Transverse sections of a stage 45 embryo. The DiI-positive cardiac NC cells are confined to the aortic sac and arch arteries (arrows) and never populate the cardiac cushions. Brightfield (A,C,E) and fluorescence (B,D,F) images are shown. AA, aortic arch; CC, cardiac cushion; OFT, outflow tract; V, ventricle.
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Hematoxylin and Eosin (H/E) staining of a transverse section through the heart of a NF stage 50 Xenopus embryo. AA, aortic arch arteries; AS, aortic sac; LA, left atrium; OFT, outflow tract; RA, right atrium; SS, spiral septum; V, [cardiac] ventricle. Scale bar: 100 μm.
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