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First published online 15 April 2009
doi: 10.1242/dev.034157

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sonic hedgehog is required in pulmonary endoderm for atrial septation

¹ Departments of Pediatrics and Pathology, University of Chicago, Chicago, IL 60637, USA.
² Department of Psychiatry, Weill Medical College of Cornell University, New York, NY 10065, USA.

View larger version (85K): [in this window] [in a new window]   Fig. 1. Hedgehog-receiving cells mark the atrial septum. (A-C) Hh-receiving lineage is marked in R26RGli1-CreERT2 embryos by a single dose of Tamoxifen (TM) at E7.5 (A), E8.5 (B) or E9.5 (C), and analyzed at E13.0. β-Galactosidase positive cells identified in whole-mount (left, 4x magnification) and cross-section (middle and right; 4x and 10x magnification, respectively) histology. In the cardiac inflow, marked cells contributed to the primary atrial septum (PAS in A-D) from embryos administered with TM at E7.5 to E9.5, the dorsal mesenchymal protrusion (DMP, right and middle) from embryos administered with TM at E8.5 to E9.5, and the atrioventricular canal endocardial cushions (blue arrowheads in A, right) from embryos administered with TM at E7.5. Marked cells are absent from the heart in embryos administered with TM at E10.5. (D) Daily administration of TM at E7.5, E8.5 and E9.5 to R26RGli1-CreERT2 embryos demonstrates that the entire PAS and DMP are populated by Hh-receiving cells, evident in caudal, mid and cranial transverse sections of an E13.0 embryo. At, atria; AS, atrial septum; L, lungs; V, ventricle.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Hedgehog-receiving cells mark the pulmonary trunk. (A-C) Hh-receiving lineage marked in R26RGli1-CreERT2 embryos by a single dose of TM at E7.5 (A), E8.5 (B) or E9.5 (C), and analyzed at E13.0. β-Galactosidase positive cells identified in whole-mount (left and middle; 4x and 10x magnification, respectively) and cross-section (right, 10x magnification) histology. In the outflow tract, marked cells contribute specifically to the pulmonary trunk (white arrowheads and PT in A-E) and pulmonary trunk endocardial cushion at each time point. Few marked cells are observed in the aorta (middle, blue arrowheads; right, `Ao'). (D,E) Comparison of E13.5 outflow tracts marked by β-galactosidase expression in R26RWnt1-Cre (D) and R26RGli1-CreERT2 (E) mice. Cells that receive a Hh signal, marked by Gli1-CreERT2, primarily populate the outer edge of the outflow tract vessels, whereas cells originating from the neural crest, marked by Wnt1-Cre, primarily populate the inner wall of the outflow tract. Gli1-CreERT2 and Wnt1-Cre expressing cells appear to populate complementary domains that may overlap slightly, but not broadly.

View larger version (79K): [in this window] [in a new window]   Fig. 3. Migration of Hedgehog-marked cells into the atrial septum. (A-E) Hh-receiving lineage marked by TM administration at E7.5 and E8.5 in R26RGli1-CreERT2 embryos and analyzed at E8.5 (A), E9.5 (B), E10.5 (C), E11.5 (D) and E12.5 (E). β-Galactosidase positive cells were identified in the cardiac inflow by whole-mount (left, 4x magnification) or section (middle and right; 4x and 10x magnification, respectively) histology. At E8.5, marked cells are observed in the dorsal mesocardium (DM). At E9.5 and E11.5, a population of marked cells extends from second heart field splanchnic mesoderm through the DM and dorsal mesenchymal protrusion (DMP) into the atria (B-D, right). At E11.5 and E12.5, the migrating Hh-marked lineage populates the primary atrial septum (PAS in D,E) and DMP (D). At, atria; AS, atrial septum; V, ventricles; Tr, pulmonary endoderm.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Migration of Hedgehog-marked cells into the pulmonary trunk. (A-E) Hh-receiving lineage marked by TM administration at E7.5 and E8.5 in R26RGli1-CreERT2 embryos and analyzed at E8.5 (A), E9.5 (B), E10.5 (C), E11.5 (D) and E12.5 (E). β-Galactosidase positive cells were identified by whole-mount (left, 4x magnification) or section (middle and right; 4x and 10x magnification, respectively) histology. At E8.5 and E9.5, marked cells are primarily outside of the heart, in the mesoderm surrounding the pharyngeal endoderm (Ph). By E10.5 and E11.5, marked cells populate the outflow tract, with a continuous population of marked cells from the pharyngeal endoderm to the outflow tract present at E10.5, and a larger proportion concentrating to the pulmonary side of the single outflow tract at E11.5. At E12.5, separate pulmonary and systemic trunks have formed and the marked cells primarily populate the pulmonary trunk. OFT, outflow tract; RV, right ventricle; PA, pulmonary artery; PT, pulmonary trunk.

View larger version (132K): [in this window] [in a new window]   Fig. 5. Atrial and atrioventricular septal defects in Gli1CreERT2 conditional Smo mutants. (A-D) E14.5 cardiac anatomy by cross-section histology in control Smofl/fl (A,C) and mutant SmoGli1-CreERT2 (B,D) embryos administered with TM at E7.5 and E8.5. Atrial septal (D, arrowhead) and atrioventricular canal (D, asterisk) defects are present in SmoGli1-CreERT2 but not Smofl/fl embryos. C and D correspond to boxes in A and B, respectively. (E,F) SmoGli1-CreERT2 embryos (F) at E10.5 show hypoplastic dorsal mesenchymal protrusions (DMP, arrow) as compared with wild-type Smofl/fl littermates (E). AS, atrial septum.

View larger version (87K): [in this window] [in a new window]   Fig. 6. Atrial septum versus atrial free wall distribution is dependent on Hedgehog signaling. (A-E) R26R;SmoGli1-CreERT2 embryos (B,D) and littermate controls (A,C) were administered with TM at E7.5 and E8.5, and dissected at E14.5 and E10.5. (A,B) E14.5 R26R;SmoGli1-CreERT2 embryos have severe atrial septal defects (asterisk), corresponding to the absence of β-galactosidase marked cells that are observed in wild-type controls. (C,D) E10.5 R26R;SmoGli1-CreERT2 embryos demonstrate a hypoplastic DMP compared with littermate controls (arrows) and an increased number of β-galactosidase positive cells in the atrial free wall (arrowheads). (E) E10.5 R26R;SmoGli1-CreERT2 embryos have significantly fewer β-galactosidase positive cells in the DMP and significantly more β-galactosidase positive cells in the atrial free wall when compared with littermate controls. AS, atrial septum.

View larger version (83K): [in this window] [in a new window]   Fig. 7. Expanded activation of Hedgehog signaling causes aberrant atrial septum development. (A-H) Constitutive activation of the Hh pathway using the Cre-activated smoothened-M2 allele (R26-smoM2) in cells that normally receive Hh signal has no effect on atrial septation, whereas activating the Hh pathway in a broader domain results in a hypercellular atrial septum. Whole-mount (A,E) and cross-section (B-D,F-G) histology. (A,B) Gli1-CreERT2 expression domain demonstrated with the R26R reporter. (C,D) Constitutive activation of the Hh pathway in the Gli1-CreERT2 expression domain has no effect on the size or overall structure of the atrial septum compared with littermate controls (arrows). (E,F) Nkx2-5Cre expression domain demonstrated with the R26R reporter. Inset in F depicts Nkx2-5Cre expression in pulmonary endoderm. (G,H) Constitutive activation of the Hh pathway in the broader Nkx2-5Cre domain results in an enlarged atrial septum when compared with wild-type littermates (arrows).

View larger version (123K): [in this window] [in a new window]   Fig. 8. Shh is required in pulmonary endoderm for atrial septation. (A-F) PtchlacZ demonstrates Hh signal responsiveness outside of the heart in two axial domains; one adjacent to the pharyngeal endoderm (Ph) and one adjacent to the pulmonary endoderm (Tr). This localization is visible at E8.5 (A,B); E9.5 (C,D); and E10.5 (E,F). (G,H) In situ hybridization against Shh in an E10.5 embryo demonstrates Shh expression in the pulmonary endoderm (blue and black arrows) and esophagus (gray arrow) during early atrial septum formation. No expression is observed in the heart. (I,J) Nkx2-1Cre and Shh expression overlap specifically in the pulmonary endoderm tissues (blue and black arrows). Removal of Shh expression from the pulmonary endoderm using Nkx2-1Cre and conditional loxP-flanked Shh (ShhNkx2.1-Cre) results in atrial septal defects (L, asterisk), which is absent from control littermates (K).

View larger version (45K): [in this window] [in a new window]   Fig. 9. Specification of cardiac progenitors by Hedgehog signaling. (A-C) Hh signaling from pulmonary endoderm and pharyngeal endoderm specifies cardiac progenitors in the posterior and anterior second heart field splanchnic mesoderm, respectively (A). Required Hh signaling occurs between E8 and E10, during heart tube and looping stages. Physical continuity between the developing atria and the posterior SHF Hh signaling, and between the outflow tract and the anterior SHF Hh signaling, is present via the dorsal mesocardium. Hh-induced cardiac progenitors migrate from the pulmonary mesoderm into the atrial septum (B), and from the pharyngeal mesoderm into the developing pulmonary trunk (C). Ao, aorta; At, atrium; LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; OFT, outflow tract; PT, pulmonary trunk.

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