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Conotruncal myocardium arises from a secondary heart field

¹ Division of Neonatology, Department of Pediatrics, PO Box 3179, Duke University Medical Center, Durham, NC 27710, USA
² Departments of Cellular Biology and Anatomy, and Pharmacology, Medical College of Georgia, Augusta, GA 30912, USA
^* Author for correspondence (e-mail:kirby013{at}mc.duke.edu )

View larger version (68K): [in a new window]   Fig. 1. Nkx2.5 and Gata-4 are expressed in the splanchnic mesoderm of the pharyngeal floor. (A) Whole-mount (stage 14) chick embryos after in situ hybridization for Nkx2.5. (B) Diagram illustrating the distal cardiac outflow tract as it is seen in A. The broken line indicates the plane of section seen in C,E. (C) Nkx2.5 mRNA can be seen in the caudal wall of the distal cardiac outflow tract (oft) and in the mesenchyme dorsal and caudal to the cardiac outflow tract (pharynx, p). (D,E) In situ hybridization showing expression of GATA-4 in the region where Nkx2.5 is expressed (arrow in D). Scale bars: 100 µm in A,C,E; 50 µm in D.

View larger version (92K): [in a new window]   Fig. 2. HNK-1 expression in the splanchnic mesoderm underlying the pharynx at stages 12 (A), 14 (B), 16 (C) and 18 (D). Over time, HNK-1 expression increases in the cranial splanchnic mesoderm and distal outflow tract and decreases in the dorsal mesocardium. (A) Section through the outflow tract (oft) showing its attachment to the ventral pharyngeal wall (p, pharynx). The splanchnic mesoderm (sm) flanking the outflow myocardium is not HNK-1 positive. (B) At stage 14, the cells in the splanchnic mesoderm flanking the primary myocardium of the outflow tract are HNK-1 positive (arrow). (C) HNK-1 expression (arrow) in the distal outflow tract myocardium is continuous with the splanchnic mesoderm caudal to the attachment of the outflow tract to the pharynx (arrowhead). (D) HNK-1 expression in an extended length of caudal outflow myocardium is continuous with that in the splanchnic mesoderm (arrowhead). (E) The myocardium of the outflow tract is positive for Slug antibody but the splanchnic mesoderm is devoid of Slug expression. Scale bars: 100 µm; A and B are the same magnification.

View larger version (55K): [in a new window]   Fig. 3. At stage 16 HNK-1-positive cells express a myocardial phenotype. Double labeling by immunohistochemistry to show the relationship of HNK-1-positive cells (green) to myocardial cells (red) in the cardiac outflow and inflow tracts. The poles of the heart exhibit a gradient of HNK-1 and myosin protein expression during looping, as seen in transverse sections of the outflow and inflow poles of a stage 16 chick embryo double labeled for myosin. (A) The distal outflow tract shows HNK-1-positive/MF20-negative cells in the splanchnic mesoderm and HNK-1-negative/MF20-positive cells in the myocardium, separated by myocardium that is positive for both HNK-1 and MF20 (yellow). (B) The splanchnic mesoderm caudal to the attachment of the outflow tract with the pharynx (arrow) is HNK-1 positive/MF20 negative. (C) The atrium displays a gradient of overlapping protein expression, similar to that seen in the outflow myocardium.

View larger version (101K): [in a new window]   Fig. 4. New outflow tract myocardium is derived from splanchnic mesoderm caudal and adjacent to the outflow. (A) Diagram of a stage 14 chick embryo showing the location of the secondary cardiac outflow field (yellow asterisk), which is located at this stage adjacent to the second arch (green). (B) Confocal image of a stage 22 chick embryo injected into the secondary heart field just caudal to the outflow tract with mitotracker at stage 14. Two new segments, a proximal fluorescent segment (asterisk) and a more distal non-fluorescent segment (star), were added to the primary tube between the time of injection at stage 14 and acquisition of this image at stage 22. The fluorescent spot (white arrow) cranial to the outflow tract shows the needle entry site for the mitotracker injection, which was originally posterior to the outflow tract. (C) The secondary heart forming region was removed from a stage 14 quail embryo and placed in the comparable site of a chick embryo at a similar stage. A transverse section at the level of the distal outflow myocardium shows two small vesicles with quail cells (QCPN antibody, brown) adjacent to the outflow tract (arrow). (D) Higher magnification of the vesicles reveals an inner lumen lined with quail cell epithelium, surrounded by MF20-positive (purple) myocardial cells. Scale bars: 100 µm.

View larger version (20K): [in a new window]   Fig. 5. Position of the outflow tract over time, relative to the aortic arch arteries. The outflow tract has a dynamic relationship with the pharynx during the looping stages. The outflow tract originally attaches ventral to arch 1 and is progressively displaced caudally to lie below pharyngeal arches 4 and 6. AA1-4, aortic arch arteries 1-4.

View larger version (68K): [in a new window]   Fig. 6. FGF-8 (A-D) and BMP-2 (E-I) expression at stages 14 and 18. (A) FGF-8 expression in the lateral pharynx. Moderate expression in arches 1 and 2 with pronounced expression in arches 3-6. FGF-8 expression persists in the pharyngeal pouch/grooves after expression in the arches has diminished. (B) Sectioned whole-mount embryo showing that FGF-8 expression in arch 3 is in the ectoderm covering the pharyngeal arches and in the endoderm where it meets the ectoderm in the pharyngeal pouches. (C) FGF-8 expression at stage 18 is diminished in the caudal pharyngeal arches and completely absent in arches 1 and 2. Expression remains in the interval between the maxillary and mandibular prominences and the second pouch/groove. (D) Sectioned stage 18 whole-mount showing the greatly diminished FGF-8 expression in arches 2 and 3. (E) BMP-2 expression in the caudal outflow tract and splanchnic mesoderm that is continuous with the outflow tract at stage 14. (F) BMP-2 expression at stage 14 stops abruptly caudal to the outflow tract in an oblique section. (G) The splanchnic mesoderm between the inflow and outflow at stage 14 is devoid of BMP-2 expression. (H) BMP-2 expression can be seen in the inflow myocardium and dorsal mesocardium at stage 14. (I) By stage 18, BMP-2 is no longer expressed by the splanchnic mesoderm or myocardium but can now be seen in the endocardium. All scale bars are 100 µm.

View larger version (82K): [in a new window]   Fig. 7. Myocardial differentiation in cultured secondary heart field is inhibited by noggin. All the panels show secondary heart fields cultured for 24 hours, incubated with BrdU for 1 hour and then doubly stained to show fluorescein-labeled antiBrdU (green) and rhodamine-labeled MF-20 (red). Medium was conditioned as indicated above the panels: A,E, unconditioned medium; B,F secondary heart field-conditioned medium; C,G, pharyngeal endoderm/ectoderm-conditioned medium; and D,H, myocardium-conditioned medium. The heart fields shown in the lower panels were cultured in the conditioned medium indicated with 15 µg/ml noggin protein added. Noggin appears to have decreased myocardial differentiation and increased proliferation, except in the secondary heart field cultured in secondary heart field conditioned medium (B,F). The endoderm/ectoderm conditioned medium caused increased proliferation in both the absence (C) and presence (G) of noggin.

View larger version (72K): [in a new window]   Fig. 8. At stage 11, the inflow tract shows continuity with the primary heart fields. (A) HNK-1 expression in the myocardium and dorsal mesocardium at the cardiac inflow tract. (B) Nkx-2.5 expression overlaps that of HNK-1 but extends slightly farther into the splanchnic mesoderm of the primary heart field. (C) Myocardial differentiation as indicated by MF-20-positive staining (red) in a stage 11 quail embryo double stained for QH-1 to show the endothelium/endocardium (green). (D) Gata-4 expression appears to replicate that of Nkx-2.5. (E) BMP-4 is not expressed in or near the cardiac inflow myocardium. (F) FGF-8 is expressed at very low levels by the ectoderm lateral to the pharynx, but is not seen in or near the cardiac inflow myocardium. All scale bars are 100 µm. Magnification in C is similar to that in B.

View larger version (41K): [in a new window]   Fig. 9. The dynamic location of events in accretion of myocardium to the outflow tract from the secondary heart field. The splanchnic mesoderm expresses Gata-4 and Nkx2.5. The outflow tract moves craniocaudally across this mesoderm. As the outflow tract approaches, Gata-4/Nkx2.5-positive cells begin to express HNK-1, move into the outflow tract, and begin to express MF20. FGF-8 expressed at the lateral sides of the pharyngeal arches may cause proliferation of the cells in the secondary heart field, while BMP-2 expressed by the distal outflow myocardium and secondary heart field near the outflow tract induce expression of the contractile proteins in the cells being incorporated into the outflow myocardium.