by Yuka Morikawa, and Peter Cserjesi Cardiac Neural Crest Expression of Hand2 Regulates Outflow and Second Heart Field Development by Yuka Morikawa, and Peter Cserjesi Circulation Research Volume 103(12):1422-1429 December 5, 2008 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Deletion of Hand2 in cNC results in multiple defects resembling CHDs. A through D, The organization of the OFT and AAA in control (A) and Hand2fx/−;Wnt1-Cre cKO (B through D) embryos at 16.5 dpc was compared using latex casts of the vessels. Figure 1. Deletion of Hand2 in cNC results in multiple defects resembling CHDs. A through D, The organization of the OFT and AAA in control (A) and Hand2fx/−;Wnt1-Cre cKO (B through D) embryos at 16.5 dpc was compared using latex casts of the vessels. B through D, cKO heart with IAA and retroesophageal right subclavian artery (rsc) (B), cKO heart with IAA and retroesophageal right subclavian artery (C), cKO heart with misalignment of right subclavian artery and pulmonary artery stenosis (D). E and F, Loss of Hand2 does not effect migration of cNC to the AAA or thymus but results in their abnormal positioning. NC cells were traced by genetic marking using β-galactosidase in Wnt1-Cre;R26R (E) and Hand2fx/−;Wnt1-Cre;R26R cKO mice (F). Embryos were analyzed for β-galactosidase expression at 15.5 dpc. da indicates ductus arteriosus; ht, heart; IAA, interrupted aortic artery; lcc, left common carotid artery; lsc, left subclavian artery; pa, pulmonary artery; rcc, right common carotid artery; rsc, right subclavian artery; thy, thymus. Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Hand2 regulates cNC expression of the semaphorin receptor PlxnA2 but not the migration of the cNC into the OFT. The expression patterns of Sema3C (A and B) and PlxnA2 (C and D) were examined in control (A and C) and Hand2fx/−;Wnt1-Cre cKO embryos (B and D) at 12.5 dpc. Figure 2. Hand2 regulates cNC expression of the semaphorin receptor PlxnA2 but not the migration of the cNC into the OFT. The expression patterns of Sema3C (A and B) and PlxnA2 (C and D) were examined in control (A and C) and Hand2fx/−;Wnt1-Cre cKO embryos (B and D) at 12.5 dpc. Expression of PlxnA2 is lost in the OFT of the cKO heart. E, Quantitative PCR analysis of Sema3C and PlxnA2. *P<0.0001. F through K, Lineage tracing of Hand2fx/+;Wnt1-Cre;R26R (F through H) and Hand2fx/−;Wnt1-Cre;R26R cKO (I through K) NC embryos at 10.5 (F and I) and 12.5 dpc (G, H, J, and K). F, G, I, and J, Whole mount staining for β-galactosidase activity. H and K, Sections through the OFT. Although the cNC populate the OFT, mutant hearts have a shortened OFT, suggesting a SHF-derived myocardial defect. Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Deletion of Hand2 in the NC results in defects of the SHF-derived RV. Control (A and C) and Hand2fx/−;Wnt1-Cre cKO (B and D) hearts were analyzed at 15.5 and 17.5 dpc. Figure 3. Deletion of Hand2 in the NC results in defects of the SHF-derived RV. Control (A and C) and Hand2fx/−;Wnt1-Cre cKO (B and D) hearts were analyzed at 15.5 and 17.5 dpc. A and B, A 17.5-dpc heart shows an increase in the size of the RV when Hand2 is deleted in the NC lineage. C and D, Transverse sections show that loss of Hand2 in the NC leads to hypertrabeculation in the RV. la indicates left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle. E, Cell proliferation rate in the trabecular zone was examined using BrdUrd labeling at 13.5 and 15.5 dpc (n=3 in each genotype and developmental stage). *P<0.0001. L indicates left ventricle; R, right ventricle. F and G, The number of cycling trabecular zone cells was determined in control (F) and cKO (G) by Ki-67 expression. Loss of Hand2 in the cNC results in a 2-fold increase in the proportion of cardiomyocytes in the cell cycle. H and I, The distribution of proliferating cells was examined in control (H) and cKO (I) RV by BrdUrd staining. Loss of Hand2 in the cNC results in an even distribution of proliferating cells in the trabecular zone. Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Expression of the trabecular marker nppa in SHF-derived trabecular myocardium is regulated by Hand2 expression in NC. A through F, The expression of nppa was examining in control (A, C, and E) and Hand2fx/−;Wnt1-Cre cKO (B, D, and F) hearts at 10.5 dpc (A and B) and 13.5 dpc (C through F) by in situ hybridization. Figure 4. Expression of the trabecular marker nppa in SHF-derived trabecular myocardium is regulated by Hand2 expression in NC. A through F, The expression of nppa was examining in control (A, C, and E) and Hand2fx/−;Wnt1-Cre cKO (B, D, and F) hearts at 10.5 dpc (A and B) and 13.5 dpc (C through F) by in situ hybridization. The level of nppa expression is decreased only in the RV of cKO hearts. At 13.5 dpc, the level of hypertrabeculation in mutant hearts has not developed to the extent as 15.5 dpc hearts (Figure 3). Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Hand2 is required for the development of RV and OFT in a cell autonomous manner. Figure 5. Hand2 is required for the development of RV and OFT in a cell autonomous manner. A through E, Hand2 was deleted in myocardium using cTnt-Cre and examined for survival and heart defects. A, Survival rate of the mutant embryos. Viability of mutant embryos begins to decline at 10.5 dpc. Morphological analysis of control (B) and Hand2fx/−;cTnt-Cre (C) hearts shows that loss of Hand2 in differentiating myocardium leads to loss of the RVs. Histological examination of hearts shows that the organization of the ventricle and outflow tract myocardium of mutant embryos (E) is disorganized and lacks trabeculation relative to control hearts (D). ao indicates aortic sac; lv, left ventricle; oft, outflow tract; rv, right ventricle; v, ventricle. Hand2 is not required for formation of the extraembryonic vasculature. F through I, Hand2 was deleted in extraembryonic and lateral mesoderm using HoxB6-Cre. The yolk sacs of Hand2fx/+;HoxB6-Cre;R26R (F and H) and Hand2fx/−;HoxB6-Cre;R26R (G and I) were analyzed at 12.5 dpc. Morphological analysis of yolk sacs shows a fully developed vasculature (F and G). Histological analysis (H and I) shows that development of membranes is not affected by loss of Hand2. vs indicates vasculature. J and K, Hand2 deletion in extraembryonic mesoderm does not cause embryonic lethality. Embryos with deletion of Hand2 in extraembryonic and lateral mesoderm survive to term. An examination of newborn Hand2fx/+;HoxB6-Cre;R26R (J) and Hand2fx/−;HoxB6-Cre;R26R (K) embryos shows that loss of Hand2 in lateral mesoderm results in severe limb defects. Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Hand2 expression in the cNC regulates multiple aspects of cardiovascular development. Figure 6. Hand2 expression in the cNC regulates multiple aspects of cardiovascular development. In the NC lineage, Hand2 plays a direct role in cardiovascular development by regulating the function of the cNC and an indirect role by regulating NE synthesis in the SNS lineages. In cNC, Hand2 regulates patterning of the AAA and OFT and formation of the ventricular septum. In addition, loss of Hand2 in the cNC affects the interaction of cNC with the SHF resulting in hyperproliferation of the SHF-derived trabecular myocardium of the RV. In addition to its role in NC during heart development, Hand2 expression in the SHF is essential for survival of RV myocardium. In the SNS, Hand2 is required for synthesis of the NE which activates adrenergic receptors and is required for embryonic survival and myocardial growth. Yuka Morikawa, and Peter Cserjesi Circ Res. 2008;103:1422-1429 Copyright © American Heart Association, Inc. All rights reserved.