Figure 6: Quail–chick chimeras reveal the contribution of preotic NCCs to developing coronary arteries.

(a) Scheme illustrating the position of the preotic NC grafted. The quail preotic NC between the midbrain and r5 was orthotopically grafted to chick embryos. (b,c) QCPN immunostaining for quail nuclei, indicating QCPN-positive cells in the septal branch (b) and papillary muscles in the right ventricle (c). QCPN-positive cells in the papillary muscle are magnified in the left lower inset (c). (d) Three-dimensional reconstruction of immunostained sections. One hundred and twenty horizontal sections of 4 μm each were used for the reconstruction. QCPN-labelled cells distribute mainly to the interventricular septum and the septal branch. (e–m) Immunohistochemistry in the septal branch with antibodies to αSMA (e) or SM22α (h,n) and QCPN (f,i,o), with merged images (g,j,p) shows the contribution of QCPN-labelled cells to SMCs (white arrows) and possibly non-muscle cells (yellow arrows). Magnified images of j reveal that QCPN-labelled nuclei are visible in the tunica media (k–m). (n–p) Immunohistochemistry in a small branch of the coronary artery with antibodies to SM22α (n) and QCPN (o), with merged images (p). Magnified images are shown in the left lower insets. (q–s) R4-Cre-labelled cells are detected by alkaline phosphatase staining. At E9.5, R4-Cre-labelled cells are specifically expressed in PA2 and the outflow tract (q). At E14.5, section staining shows their expression in the conal region (r). Boxed area in r is magnified in s, a coronary artery is covered with R4-Cre-labelled cells. AV, aortic valve; PV, pulmonary valve; CA, coronary artery; OFT, outflow tract; PA2, second pharyngeal arch; PM, papillary muscle; RV, right ventricle; LV, left ventricle; SB, septal branch; scale bars, 500 (q), 200 (r), 100 (c), 10 (b) and 40 μm (e–j,n–p).