Laterality of the internal organs of vertebrates is determined by asymmetric Nodal signalling in the lateral plate mesoderm1. A deficiency of such signalling results in heterotaxia syndrome, characterized by anomalous laterality of visceral organs and complex congenital heart conditions1. Pitx2, the transcription factor induced by the Nodal signal, regulates left–right asymmetric morphogenesis1,2,3,4. The cellular and molecular bases of asymmetric morphogenesis remain largely unknown, however. Here we show that ablation of unilateral Pitx2 expression in mice impairs asymmetric remodelling of the branchial arch artery (BAA) system, resulting in randomized laterality of the aortic arch. Pitx2-positive cells were found not to contribute to asymmetrically remodelled arteries. Instead, Pitx2 functions in the secondary heart field5 and induces a dynamic morphological change in the outflow tract of the heart, which results in the provision of an asymmetric blood supply to the sixth BAA. This uneven distribution of blood flow results in differential signalling by both the platelet-derived growth factor receptor and vascular endothelial growth factor receptor 2. The consequent stabilization of the left sixth BAA and regression of its right counterpart underlie left-sided formation of the aortic arch. Our results therefore indicate that haemodynamics, generated by a Pitx2-induced morphological change in the outflow tract, is responsible for the asymmetric remodelling of the great arteries.
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We thank S. Iseki, A. McMahon and H. Sucov for Wnt1-Cre mice; the staff of MRC Technology in Edinburgh for technical assistance with OPT; K. Yamashita for transgene construction; K. Mochida, S. Ohishi and Y. Ikawa for general technical assistance; Primetech Corp. for technical assistance with Vevo770; and P. Soriano, T. Kubo, K. Ozono, T. Sano, T. Matsusita, S. Kogaki and the cardiologists in the Department of Pediatrics, Osaka University Medical School, for advice. This work was supported by grants (to H.H.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by CREST.
Author Contributions Project planning was mainly performed by K.Y.; most of the experiments were carried out by K.Y. and the remaining experiments by H.S. and H.H.; the manuscript was written by K.Y. and H.H.
The authors declare no competing financial interests.
This file contains Supplementary Discussion 1-2, Supplementary Tables S1-S2, additional references, Supplementary Figures S1-S7 with Legends and Supplementary Videos 1-9 Legends (PDF 1435 kb)
The file contains Supplementary Video 1 which shows the appearance of a control (unligated) cultured embryo. This movie shows that the viability of the control embryos in the artery ligation experiment could not be affected. (MOV 849 kb)
The file contains Supplementary Video 2 which shows the appearance of a ligated cultured embryo. This movie shows the ligation of the left branchial arch arteries could not affect the viability of the embryos. (MOV 316 kb)
The file contains Supplementary Video 3 which shows the blood supply in the left BAAs of the control embryo shown in Supplementary Video 1. This movie shows the intact blood supply into the left side branchial arch arteries in the unligated embryos. (MOV 2346 kb)
The file contains Supplementary Video 4 which shows the loss of blood supply in the left BAAs of the ligated embryo shown in Supplementary Video 2. This movie shows the loss of blood supply into the left side branchial arch arteries in the ligated embryos. (MOV 1564 kb)
The file contains Supplementary Video 5 which shows the appearance of a control embryo cultured with vehicle (DMSO) alone. This movie shows that the culture condition without the inhibitors could not affect the viability of the embryos (MOV 560 kb)
The file contains Supplementary Video 6 which shows the appearance of an embryo cultured with VEGFR inhibitor (2 μM) and AG1296 (50 μM). This movie shows that the culture condition with VEGFR inhibitor and AG1296 could not affect the viability, the appearance, and the major vessels of the embryos. (MOV 548 kb)
The file contains Supplementary Video 7 which shows the appearance of an embryo cultured with AG1433 (10 μM).This movie shows that the culture condition with AG1433 could not affect the viability, the appearance, and the major vessels of the embryos. (MOV 573 kb)
The file contains Supplementary Video 8 which shows the appearance of an embryo cultured with propranolol (10 μM).This movie shows that propranolol (10μM) could reduce the heart rate significantly. (MOV 657 kb)
The file contains Supplementary Video 9 which shows the appearance of an embryo cultured with propranolol (25 μM). This movie shows that the bradicardia induced by propranolol (25μM) was more significant than by propranolol (10M). (MOV 892 kb)
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Yashiro, K., Shiratori, H. & Hamada, H. Haemodynamics determined by a genetic programme govern asymmetric development of the aortic arch. Nature 450, 285–288 (2007). https://doi.org/10.1038/nature06254
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