Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

Abstract

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357-372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.

Figure 1

Expression of hedgehog signaling components during secondary heart field (SHF) development in chick embryos. (A, A′) Shh in situ hybridization at HH 15 shows expression in pharyngeal clefts 2 and 3 and the foregut (A), specifically the pharyngeal endoderm (PE, A′). (B, B′) Ptc2 in situ hybridization at HH 16 shows a similar wholemount pattern (B); however, Ptc2 is restricted to the SHF mesoderm adjacent to the Shh-expressing endoderm. (C, C′) Within 12 hours, cyclopamine (CPA)-treated embryos show no expression of receptor and downstream target Ptc2, as observed by in situ hybridization. Embryos were treated at HH 14 and collected at HH 16. Ptc2 expression is absent after CPA treatment. Time allowed for color development was the same for embryos in both B and C. (D) Quantitative RT-PCR was used to analyze isolated SHF from HH 16 embryos. Both Ptc1 and Ptc2 are significantly down-regulated after CPA treatment (p<0.01 for both). Scale bar in C is 100 μm in A–C, and the scale bar in C′ is 200 μm in A′ and 100 μm in B′–C′; OFT, outflow tract. Scale bar in B is 100 μm and applies to A; scale bar in D is 100 μm and applies to C.

Figure 2

Cyclopamine (CPA) reduces proliferation and migration in vitro. (A) Before and (B) after images of a control explant during the 16-hour migration assay. The perimeter of the explant is highlighted in red. (C, D) Explants were treated with PBS (C) or CPA (D) and labeled with BrdU (green) and DAPI (blue). Note the reduction of BrdU-positive secondary heart field (SHF) mesoderm after CPA treatment. (E) Treating SHF explants with CPA reduces proliferation after 24 hours. (F) Treating SHF explants with CPA reduces migration over a 16-hour period. The highest dose reversed the anti-proliferative effect, suggesting a narrow range of anti-proliferative signaling. Scale bar is 25 μm in A and B. *p ≤ 0.05, **p ≤ 0.0001

Figure 3

Cyclopamine (CPA) treatment disrupts secondary heart field (SHF) proliferation in ovo. (A) HH 16 control embryo and (B) HH 16 embryo treated with 0.6 μg/μl CPA at HH 14. Embryos were labeled with BrdU (green nuclei) at HH 16; myocardium is red. (C) Total BrdU-positive cells counted in 10-cell increments at HH 15 to 18. SHF cells were counted starting at the myocardial border of the outflow tract (OFT; increment 1), as delineated with a myocardial marker (red) in (A) and (B), and moving caudally through the splanchnic mesoderm (with increment 8 being the most caudal). Between HH 15 and 17, proliferation in the SHF adjacent to the pharyngeal endoderm (the caudal-most 30 cells) occurs at nearly twice the rate that is seen in the 50 cranial-most cells. Within 12 hours of cyclopamine treatment (HH 16), proliferation is no longer increased in the caudal SHF (p<0.01 for increments 6–8); this decrease is maintained through HH 17 (p<0.05 for increments 6–8). By HH 18, no differences are observed in CPA-treated embryos compared to control embryos. Scale bar is 100 μm.

Figure 4

Cyclopamine (CPA) treatment induces arterial pole defects. (A) PBS-treated control; (B), (D), hearts treated with 0.6 μg/μl CPA at HH 14; (C) heart treated with 1.0 μg/μl CPA at HH 18. Embryos treated at HH 14 show a range of defects, but all have a single outflow vessel, and the origins of the great arch arteries are shifted to the right side and originate more proximal to the ventricles. Histological analysis identified the single vessel. The single vessel in (B) is an Ao (Ao); the single vessel in (D) is persistent truncus arteriosus (PTA). Embryos treated at HH 18 (C) are morphologically indistinguishable from controls. PT, pulmonary trunk; RB, right brachiocephalic; LB, left brachiocephalic. Scale bar is 175 μm.

Figure 5

Histological analysis of HH 35 hearts. (A–A″) PBS-treated control; (B–B″; C–C″) heart treated with 0.6 μg/μl CPA at HH 14; (D–D″) heart treated with 1.0 μg/μl at HH 18. In the control heart (A), the aortic vestibule (AoV) and pulmonary infundibulum (PI) are separated (A) and about the same size. At the valve level, two robust arterial trunks can be seen in the correct orientation. The aorta (Ao) is identified based on its origin from the AoV (A), its posterior position relative to the pulmonary artery (P) at the valve level (A″), and the presence and position of two coronary artery stems from the coronary sinuses (arrows in A″). After CPA treatment at HH 14 (B), a ventricular septal defect allows a connection between the small PI and the AoV (B). The PI disappears before reaching the single outflow vessel (B″). The presence of two coronary arteries penetrating the single outflow vessel further confirms the identity of the Ao (arrows in B″), indicating that this embryo had pulmonary atresia. Another embryo treated at HH 14 (C) exhibits pulmonary atresia. While a ventricular septal defect is present, this defect is very high. The single outflow vessel has five valve leaflets (C″); in addition, two coronary arteries penetrate the aortic side of the single outflow vessel, one of which is seen in C″ (arrow). Treatment at HH 18 (D) results in a well-formed ventricular septum (D), divided arterial valves, (D″) and normal coronary arteries (arrows in D″). Scale bar is 250 μm in A–A″ and 100 μmin A″; the same scales apply to rows B–D.

Figure 6

India ink injections of PBS- (A, C, E) and cyclopamine (CPA)-treated (B, D, F) embryos. CPA-treated embryos at HH 18 (B) have a persistent first arch artery, compared to the control (A), where it has begun to regress. In addition, the junction between the aortic sac and the outflow tract is widened after CPA treatment. By stage HH 22-23, arch arteries 3, 4, and 6 are normally open (C); however, after CPA treatment, pharyngeal arch arteries 4 and 6 are very small (thin endothelial strands marked with arrows in D). Right arch arteries 4 and 6 are so thin that the left arch arteries are visible, unlike in the control. At HH 29, the appropriate pharyngeal arch arteries appear to be present (compare cyclopamine-treated F with control E). Arch arteries are labeled according to arch number and side. Scale bar is 100 μm.

Figure 7

Sectioned India ink-injected embryos at HH 29. A–D, PBS-treated embryo; EH, CPA-treated embryo. In the control, the left fourth arch artery (L4) is regressing (B), and a septum is beginning to divide the outflow tract (D). In the CPA-treated embryo, the left 4/6 arch artery (L4/6) is not at the level of either the fourth or sixth arch (E–F), and an open communication persists between the pulmonary (P) and aortic (A) trunks (arrow, H). Arch arteries are labeled according to arch number and side. Scale bar is 200 μm.

Figure 8

Quail-chick chimeras treated with PBS (A, C, E) and cyclopamine (CPA, B, D, F) show that the cardiac neural crest enter and septate the outflow tract (OFT). Apoptosis was analyzed before septation (HH 23-24, A, B). In both control (A) and cyclopamine-treated (B) chimeras, the quail neural crest-derived cells (green) are not TUNEL-positive (red) as they enter the OFT, indicating that these cells are not undergoing apoptosis. Nuclei are counterstained with DAPI (blue). Of note, the aortic outlet (AO) is open in both control (A) and CPA-treated (B) embryos; the pulmonary outlet (PO), though, is only open in the control embryo (A). After septation occurs (HH 35, C–F), the presence of quail neural crest-derived cells in the outflow tract was confirmed (red in C and close-up E; green in D and close-up F), and embryos were co-labeled with either myocardial marker MF20 (red in D and F) or smooth muscle marker SM22-α (green in C and E). Quail neural crest-derived cells are still present after CPA treatment (D); however, these cells do not form two equal-sized outflow vessels as seen in the control (C). Instead, the prominent outflow vessel in the CPA-treated chimera is the aorta (A), as determined by connection to the ventricles and the presence of a coronary artery; a small pulmonary (P) is septated by the quail neural crest-derived cells (highlighted with an arrow in D). The close-ups in E and F suggest that there may be a cell size difference after CPA treatment. C–F are co-labeled with DAPI. Scale bar is 100 μmin A–D and 50 μm in E–F.