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First published online 7 March 2007
doi: 10.1242/dev.02824

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Independent requirements for Hedgehog signaling by both the anterior heart field and neural crest cells for outflow tract development

¹ Departments of Cell Biology and Pediatrics, Neonatal-Perinatal Research Institute, Duke University Medical Center, Durham, NC 27710, USA.
² Baylor College of Medicine, Houston, TX 77030, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Nkx2.5Cre/+; Shhflox/- specifically ablates pharyngeal endodermal SHH and results in a failure of OFT septation. (A) Shh is detected by in situ hybridization in the pharyngeal endoderm (arrowheads) at E10.5. (B) Nkx2.5Cre expression, as detected by R26R, demonstrates overlap with Shh expression in the pharyngeal endoderm alone (arrowheads). (C,D) Conditional ablation of Shh in E10.5 Nkx2.5Cre/+; Shhflox/- embryos results in the specific loss of pharyngeal endodermal Shh (arrow, C versus D), but not of notochord and floorplate Shh expression, as expected (arrowheads, C,D). (E-F') Ptch1lacZ expression at E10.5 demonstrates a specific loss of Hh activity in the pharyngeal endoderm and overlying mesoderm and mesenchyme (arrows, F,F') while maintaining other domains of expression, such as in the floorplate of the neural tube (arrowheads, E-F'). (G-H') Wild-type and Nkx2.5Cre/+; Shhflox/- E18.5 ink injections (G,H) demonstrate a single OFT vessel in mutants (arrow in H), whereas section analysis (G',H') reveals a complete atrioventricular canal defect (arrows in H'). In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos. B; brachiocephalic artery; LCC, left common carotid; LSub, left subclavian; OFT, outflow tract; RCC, right common carotid; RSub, right subclavian.

View larger version (96K): [in this window] [in a new window]   Fig. 2. OFT lengthening and conotruncal cushion development is abnormal in Nkx2.5Cre/+;Shhflox/- embryos due to cell death. (A-D) Comparing E10.5 OFT and right ventricle length between wild-type (A,C) and Nkx2.5Cre/+; Shhflox/- (B,D) embryos reveals a reduction in length of the mutant OFT and right ventricle (all bars are identical in adjacent images and quantified in G), whereas the left ventricle is unchanged. Paired t-test P=0.002 for OFT and P=0.025 for the right ventricle. (E-F') OFT conotruncal cushions (F,F', arrows) are reduced at E10.5 compared with Nkx2.5Cre/+; Shhflox/+ (E,E'). Immunohistochemistry markers in E and F are PECAM (red) and AP2 (green). (H-K'') Levels of cell death, as revealed by Lysotracker Red analysis, at E10.5 (H-I', sagittal views; J-K'', frontal views) demonstrate increased cell death in the pharyngeal endoderm (K,K'), in the splanchnic mesoderm (I,K') and within the core-arch mesoderm (I') of mutants. In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos. A, atrium; OFT, outflow tract; PE, pharyngeal endoderm; SM/NCC, splanchnic mesoderm/neural crest cells; V, ventricle; PS, pharyngeal space; 1-4, pharyngeal arch 1-4.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Cardiovascular, but not other neural crest-dependent, derivatives are affected by loss of endodermal Shh. Comparison of wild-type, Nkx2.5Cre/+; Shhflox/- and Shh-/- embryos reveals that only certain NCC-derived structures depend on endodermal Shh. (A-F) Cranial nerves (CN) are relatively normal in Nkx2.5Cre/+; Shhflox/- embryos (B) compared with Shh-/- mutants (C), whereas their arch-artery patterning (E) more closely resembles Shh-/- defects (F) rather than wild-type (A,D). (G-O) Similarly, expression of the NCC markers Ap2 and CrabP1, as well as AP2 antibody, in Nkx2.5Cre/+; Shhflox/- mutants (H,K,N) resemble wild-type (G,J,M) and not the abnormal pattern seen in Shh-/- embryos (I,L,O). In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos.

View larger version (104K): [in this window] [in a new window]   Fig. 4. Ablation of Hh signaling within CNCCs results in a single OFT due to a reduced number of CNCCs. (A,B) Wnt1-Cre-mediated ablation of Smo results in a single OFT and abnormal arch-artery patterning at E18.5 (B, arrows) compared with controls (A). (C-D') NCC-lineage trace using the R26R reporter demonstrates a reduction in the total number, as well as abnormal localization, of CNCCs within the developing OFT at E10.5 (D,D', arrows) compared with controls (C,C'). (C'',D'') Section analysis of C and D in the frontal plane reveals reduced cushion size (D'') compared with controls (C''). (E-F') Reduction of Hh signaling is detected by the Ptch1lacZ activity shown in whole-mount (F) and sagittal (F') sections compared with controls (E,E'). In Wnt1-Cre; Smoflox/- mutants, there is decreased activity within the pharyngeal arches (bracket, F) and dorsal to the aortic sac (arrow in F). (G,H) Constitutive activation of the Hh pathway within NCCs using SmoOEX also results in a single OFT (H versus G). (I-L') Proximal/distal section analysis of Wnt1-Cre; SmoOEX embryos at E10.5 (J,J') and E11.5 (L,L') reveals abnormal localization and the compaction of cells within the OFT (J,J'), and that, despite the presence of cushions, septation is not taking place (L,L') when compared with controls at E10.5 (I,I') and E11.5 (K,K'). In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos. Ao, aorta; Pa, pulmonary artery; AS, aortic sac; AHF, anterior heart field; A, atrium; V, ventricle.

View larger version (52K): [in this window] [in a new window]   Fig. 5. AHF ablation of Hh signaling results in a single OFT due to septation failure, but not in a shortened OFT. (A,B) Ink injections of wild type (A) and AHF-Cre; Smoflox/- (B) at E18.5 reveals a single OFT in the mutants. (C-F) ß-galactosidase staining in AHF-Cre; Smoflox/- mutants that are also transgenic for Tie2-lacZ reveals that the OFT is not significantly shortened (D,D') and that cushions form (D',F) similar to control (C,C',E) embryos (bar length is identical in C,D). Arrowheads point to cushions compressing the endothelial channel in C',D'. (E,F) Section analysis of E10.5 AHF-Cre; Smoflox/- mutants also transgenic for the Cre reporter R26R (F) reveals that CNCCs populate the OFT cushions in relatively normal numbers compared to controls (E). (G-H') Although the cushions are well-formed at E12.5 (H,H', arrows), septation does not complete in AHF-Cre; Smoflox/- mutants, resulting in a single OFT channel (H,H') compared with controls (G,G'). (I,J) Ptch1lacZ expression in AHF-Cre; Smoflox/- mutants reveals a specific loss of expression within the AHF, including in the expression entering the OFT (J versus I, arrow). In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos. Ao, aorta; Pa, pulmonary artery; OFT, outflow tract; RA, right atrium; LV, left ventricle.

View larger version (87K): [in this window] [in a new window]   Fig. 6. Endodermal Tbx1, Fgf8 and Bmp4 expressions appear normal in mutants. (A-C) In situ analysis for Tbx1 at E9.5 in Nkx2.5Cre/+; Shhflox/- (B) and Shh-/- (C) mutants appears normal within the pharyngeal endoderm when compared to control (A). (D-F,I,J) Similarly, mRNA levels for Fgf8 (D-F) and Bmp4 (I,J) also appear normal. (G-H') Fgf8lacZ expression is also grossly normal in Shh-/- pharyngeal endoderm (H) and OFT/right ventricle (H') when compared to controls (G,G').

View larger version (35K): [in this window] [in a new window]   Fig. 7. Model of Hh signaling during OFT development. Pharyngeal-derived (PE) SHH performs three main roles. First (1), SHH acts as a direct survival factor to CNCCs. Second (2), in AHF-derived myocardium, SHH acts via Smo to provide a patterning signal that is required for an unknown function in completing septation between E10.5 and E12.5. Third (3), SHH is a direct survival factor for the pharyngeal endoderm, and its loss results in the absence of a secondary signal ("X") necessary for AHF survival and for the lengthening of the OFT. Disruption of any of these processes either alone or in combination can result in a single-OFT phenotype, via different mechanisms. RV, right ventricle.