Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries

Abstract

Rationale: The genes encoding fibroblast growth factor (FGF) 8 and 10 are expressed in the anterior part of the second heart field that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects.

Objectives: Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole.

Methods and results: We generated mesodermal Fgf8; Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance; however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to pharyngeal arch artery phenotypes and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches.

Conclusions: These results show functional overlap of FGF8 and FGF10 signaling from second heart field mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during pharyngeal arch artery formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.

Figure 1

OFT and RV morphology of E9.5 Fgf8;Fgf10;MesP1Cre compound mutants. (A–L) Right lateral views of embryos are shown, and genotypes are listed above the panels. In Fgf8^flox/+ (A), Fgf8^flox/+;Fgf10^+/− (B), and Fgf8^flox/+;Fgf10^−/− (C) embryos, OFT and RV morphology is normal. In Fgf8^flox/flox (D–F), Fgf8^flox/flox;Fgf10^+/− (G–I) and Fgf8^flox/flox;Fgf10^−/− (J–L) embryos, the angle between the proximal and distal regions of the OFT is more obtuse (lines) or, in severe cases, absent, and/or the RV is smaller (circle) when compared with normal hearts (A–C). (M–O) Sections of the OFT of E10.5 embryos with the genotypes indicated (M–O). CNC has invaded the OFT (arrowheads), and the endothelium is beginning to undergo epithelial-mesenchymal transformation (arrows) where the cushions will form in Fgf8;Fgf10 double heterozygous control embryos (M). In Fgf8;Fgf10 mutant embryos, these processes are compromised (N,O) and most notably in the double homozygous mutant, the OFT is smaller and misshapen (O). Pharyngeal arches are numbered in A. Scale bar, 200µm.

Figure 2

Reduction of FGF dosage affects morphology and gene expression in the OFT and RV. (A) Phenotype of OFT and RV morphology of E9.5 Fgf8;Fgf10;MesP1Cre compound mutants are divided into normal, mild, moderate and severe according to OFT length, the angle between the proximal and distal regions of the OFT, RV size and looping, based on blind tests. Increasingly severe phenotypes (see Figure 1) are observed as Fgf gene dosage is reduced. Numbers of embryos (n) are indicated below each column. (B) Quantitative PCR analysis of transcripts for FGF signaling effectors, Pea3 and Erm, and for Isl1, normalized to GAPDH transcripts, in extracts from the pharyngeal region dissected from six embryos with the genotypes indicated at 24 to 26 somite stages. Results are shown relative to that for Fgf8;Fgf10 double heterozygotes. Error bar indicates the standard deviation.

Figure 3

PAAs in Fgf8;Fgf10;MesP1Cre compound mutants. (A–L) PAAs are visualized by ink injection at E10.5. Genotypes are listed above each panel. At E10.5, 3rd, 4th and 6th PAAs are observed in the arches on both sides of the embryo. Fgf8^flox/+, Fgf8^flox/+;Fgf10^+/−, and Fgf8^flox/+;Fgf10^−/− embryos have normal PAA patterns (A–C), while Fgf8^flox/flox, Fgf8^flox/flox;Fgf10^+/− and Fgf8^flox/flox;Fgf10^−/− embryos have various defects (arrows in D–L), including missing 3rd, 4th, and 6th PAAs, and retention of the 2nd PAA. (M) Summary of ink injection results for each embryo examined. Black columns represent PAAs labeled by the ink, white columns represent PAAs where ink labeling was negative, and gray columns represent PAAs which were weakly labeled. (N,O) Sections of embryos at E10.0 in the region of the pharyngeal arches, with the genotypes indicated. In control Fgf8;Fgf10 double heterozygous embryos, the 2nd , 3rd, and 4th arches are evident (N, arrows). The 4th pharyngeal pouch (4p) is also visible. In the Fgf8;Fgf10 double homozygous mutant sections shown, the 4th arch is present but the PAA is not detectable on the right-hand side. On the left side, the 3rd PAA is missing (O). DA, dorsal aorta; as, aortic sac. Scale bar, 200µm.

Figure 4

Heart and PAA defects in Fgf8;Fgf10;MesP1Cre compound mutants at later stages (E15.5–E18.5). Genotypes are listed above each panel. (A–C) Heart and PAAs have normal structures in Fgf8^flox/+ and Fgf8^flox/+;Fgf10^+/− embryos. In Fgf8^flox/+Fgf10^−/− and Fgf8^flox/flox;Fgf10^−/− embryos (D,K–M), in the absence of FGF10, the position of the apex of the heart is random, and the pulmonary arteries are absent; other parts of the Fgf8^flox/+;Fgf10^−/− heart and the PAAs are normal (D). (E–M) Fgf8^flox/flox (E–G), Fgf8^flox/flox;Fgf10^+/− (H–J) and Fgf8^flox/flox;Fgf10^−/− (K–M) embryos have PAA and heart defects. As examples of PAA defects, E, H and K show external and internal carotid arteries (ECA, ICA) directly arising from the aortic arch, F, I and K show an aberrant origin of the right subclavian artery (ARSA), and an abnormal right aortic arch (RtAA) is also observed (I). Heart defects include abnormal alignment of the OFT (transposition of the great arteries, TGA; double outlet right ventricle, DORV) (F,J,L), and ventricular septal defects (VSD) (G,J,M). Ao, aorta; PT, pulmonary trunk; RA and LA, right and left atria; RCC and LCC, right and left common carotid arteries; LSA, left subclavian artery; BT, brachiocephalic trunk; RV and LV, right and left ventricles; VS, ventricular septum; TrV, tricuspid valve; AoV, aortic valve; PV, pulmonary valve.