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First published online 2 October 2008
doi: 10.1242/dev.025437

Development 135, 3599-3610 (2008)
Published by The Company of Biologists 2008
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An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart

Eon Joo Park1,*, Yusuke Watanabe2,*, Graham Smyth3, Sachiko Miyagawa-Tomita4, Erik Meyers3, John Klingensmith3, Todd Camenisch5, Margaret Buckingham2 and Anne M. Moon1,6,7,{dagger}

1 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84112, USA.
2 Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, Paris 75015, France.
3 Department of Cell Biology, Duke University, Durham, NC 27710, USA.
4 Department of Pediatric Cardiology, Tokyo Women's Medical University, Tokyo 162-8666, Japan.
5 Department of Pharmacology and Toxicology, University of Arizona, Tuscon, AZ 85721, USA.
6 Department of Pediatrics, University of Utah, Salt Lake City, UT 84112, USA.
7 Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT 84112, USA.


Figure 1
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  		Fig. 1. Mouse OFT cushion morphogenesis is disrupted in Fgf8c/-;Isl1Cre mutants. Fgf8c/-;Isl1Cre mutants are labeled as Fgf8;Isl1Cre. (A) Transverse section across the proximal outflow tract (OFT) of a E10.5 control embryo shows many mesenchymal cells in a thick layer of cardiac jelly (bracket) lining the myocardium (My). The arrow points from proximal (close to right ventricle) to distal OFT. (B) Sagittal section spanning the OFT and atrioventricular canal (AVC) cushions of E10.5 control embryo. (C) Transverse section across the proximal OFT of an E10.5 Fgf8;Isl1Cre mutant embryo reveals hypoplastic cushions (arrowheads) with few mesenchymal cells in a thin layer of cardiac jelly (bracket). (D) Sagittal section of the OFT and AVC of an E10.5 Fgf8;Isl1Cre mutant with hypocellular OFT cushions (arrowhead) but normal AVC cushions. (E,F) Sagittal sections of an E11.5 control embryo shows mesenchymal cells throughout the OFT cushions. (G,H) Sagittal sections of E11.5 Fgf8;Isl1Cre mutants with hypoplastic proximal (black arrowhead) and distal (green arrowhead) OFT cushions. (I) Section through the heart (ht) of an X-Gal-stained 6-somite stage (6ss) Rosa26lacZ;Tie2Cre embryo. (J) Section of proximal OFT cushion (arrowhead) of an E11.5 Rosa26lacZ;Tie2Cre embryo. All of the cells in the proximal cushion are stained and the distal cushion contains unstained CNC. (K) Section of an E9.25 Rosa26lacZ;Tie2Cre embryo. Box encloses proximal OFT cushion. (L) Mesenchymal cells that invaded a collagen gel from an E9.25 Rosa26lacZ;Tie2Cre proximal OFT explant (box in K). For quantitated data, see Fig. S1 in the supplementary material.

 

Figure 2
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  		Fig. 2. Expression of Spry2-IRES-alkaline phosphatase in the mouse second heart field using Mesp1Cre. (A-D) Alkaline phosphatase activity is absent from control sprouty 2 gain-of-function (Spry2-GOF) (A,C), but present in the second heart field (SHF, white bracket) and heart tube in Spry2-GOF;Mesp1Cre (B,D) embryos at E9.5. Pharyngeal arches are numbered. (E-H) In situ hybridization for Spry2 mRNA at E8.5 in control (E,G) and Spry2-GOF;Mesp1Cre (F,H) embryos, on whole-mounts (E,F) and transverse sections (G,H). White brackets indicate SHF; arrow in E points to the OFT myocardium; arrows and arrowheads in G,H indicate the OFT and unlabeled endothelial cells, respectively. (I-J') Immunochemistry on E8.5 whole-mount (I,J) and sectioned (I',J') control (I,I') and Spry2-GOF;Mesp1Cre (J,J') embryos shows decreased phosphoERK1/2 (pERK) staining in the SHF of mutants (white brackets in whole-mounts, red arrowheads in sections). Pharyngeal arch 1 is numbered. (K,L) In situ hybridization shows expression of Erm in right-sided whole-mount views of control (K) and Spry2-GOF;Mesp1Cre (L) embryos at E9.5. White brackets indicate SHF; arrows in L point to epithelial expression domains that are not affected (as expected); arrowhead in L indicates decreased somite expression.

 

Figure 3
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  		Fig. 3. Wild-type OFT myocardium rescues the endothelial-to-mesenchymal transformation defects of Fgf8;Isl1Cre mutants. (A) Explant (Ex) culture of mouse E9.5 control OFT. (A') Close-up of boxed region in A. Arrowhead demarcates a rare, rounded cell that migrated from the explant but failed to invade. (B) Explant culture of an E9.5 Fgf8;Isl1Cre mutant OFT reveals failure of endothelial EMT. (B') Close-up of boxed region in B. Arrows point to rare cells that underwent EMT. (C,C') Co-culture of E9.5 OFTs from Rosa26lacZ;Isl1Cre (blue) and controls (white). (C') Enlargement of boxed area in C. (D,D') Co-culture of control and Fgf8;Rosa26lacZ;Isl1Cre E9.5 OFTs shows rescue of mutant cells (blue, arrowheads). (D') Enlargement of boxed area in D. (E,E') Complementary experiment to D; control cells are β-galactosidase-positive (blue) and rescued Fgf8 mutant cells (arrowheads) are white. (E') Enlargement of boxed area in E. For quantitated data, see Fig. S2 in the supplementary material.

 

Figure 4
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  		Fig. 4. OFT remodeling is sensitive to Fgfr1/2 gene dosage in mesodermal OFT precursors and pharyngeal endoderm. Whole-mount thorax dissections (A-H) and sectioned preparations (A'-H') of mouse embryos at E18.5. R1, Fgfr1; R2, Fgfr2. (A,A') Double homozygous conditional control with normal relationships of atria (LA, left atrium; RA, right atrium), ventricles (LV, left ventricle; RV, right ventricle), aorta (Ao), main pulmonary artery (PA), pulmonary valve (PV), right and left subclavian arteries (RSA, LSA) and right and left common carotid arteries (RCC, LCC). The normal shape of the RV conus (co) is indicated by the dashed line in E. These anatomic annotations are used in this and all subsequent figures. (B,B') Normal Fgfr1c/+;Fgfr2c/c;Mesp1Cre OFT. (C,C') Fgfr1c/c;Fgfr2c/+;Mesp1Cre embryo with the DORV alignment defect (double arrowhead). Hypoplasia of the RV conus (dashed line) causes the aortic valve (AV) and PV to be abnormally located in the same plane (C'). The aortic arch is interrupted (arrowhead). (D,D') Fgfr1c/c;Fgfr2c/c;Mesp1Cre embryo with type III PTA: the aortic arch and left and right branch pulmonary arteries (LPA, RPA) arise from the unseptated truncus arteriosus (TA). The truncal valve (TV) is committed to the RV (arrowhead). ca, coronary artery. (E,E') Normal OFT in an Fgfr1c/+;Fgfr2c/+;Isl1Cre double heterozygote. (F,F') Fgfr1c/+;Fgfr2c/c;Isl1Cre mutant with conal hypoplasia (dashed line) and misaligned Ao and PA. (G,G') Fgfr1c/c;Fgfr2c/+;Isl1Cre, type I PTA with pentacuspid TV (G') and interrupted aortic arch (arrowhead). (H,H') Fgfr1c/c;Fgfr2c/c;Isl1Cre mutants have type III PTA. This TV is bicuspid and arises from the RV (H').

 

Figure 5
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  		Fig. 5. Antagonism of FGF signaling by overexpression of Spry2 disrupts OFT development. (A-I) Isolated E18.5 mouse hearts from controls (A-C) and Spry2-GOF;Mesp1Cre mutants (D-I). (A) Frontal view. (B) Superior view, ventral surface at top; the AV is dorsal and right of the PV. trv, tricuspid valve; mv, mitral valve. (C) Frontal view; RV wall removed to show PV and ventricular septum (VS). (D) Frontal view of type III PTA. (E) Superior view of embryo in D. (F) Frontal view of membranous VSD (arrowhead). (G) Superior view showing a bicuspid aortic valve (BAV, arrowhead). (H) Superior view of mutant with DORV; probe (black line) passes abnormally from the Ao into the RV. (I) Superior view, atria removed, of atrioventricular septal defect (AVSD) with incomplete fusion of the AVC cushions. (J-O) mRNA in situ hybridizations of E9.5 control (J,L,N) and Spry2-GOF;Mesp1Cre mutant (K,M,O) embryos. Mutant OFTs are short and aberrantly angulated (white lines) and the RV is hypoplastic. (J,K) Wnt11 transcripts are decreased in the mutant OFT. (L,M) Bmp4 transcripts are decreased in the mutant OFT (arrowhead) and in the SHF (arrow). (N,O) Crabp1 expression is decreased in mutant pharyngeal arch neural crest (arrowheads). (P,Q) Sections of E10.5 control (P) and Spry2-GOF;Mesp1Cre mutant (Q). Arrowhead points to reduced CNC and arrow to proximal cushion defects in the mutant. (R,S) Explant cultures of control (R) and Spry2-GOF;Mesp1Cre (S) OFTs show reduction in EMT when reception of FGF signaling in mesodermal cells is compromised. (T,U) Immunohistochemical detection of phosphohistone H3 (pHH3, red) and Isl1 (green) in E8.5 [11-somite stage (ss)] embryos. (V) Quantitation of pHH3+ cells as a percentge of the total number of cells in the SHF and OFT of E8.5 (10-11ss) control and Spry2-GOF;Mesp1Cre mutants. Proliferation is significantly decreased in both tissues (SHF, P=0.039; OFT, P=0.05). Three sections per embryo were counted and n=3 embryos per genotype. The s.d. was measured with Student's t-test.

 

Figure 6
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  		Fig. 6. Loss of FGF8 signaling in the SHF and pharyngeal endoderm disrupts BMP and TGFβ signaling. (A,B) Intensity maps of relative expression of members and targets of the FGF and BMP/TGFβ signaling pathways obtained from four Agilent microarrays comparing Fgf8;Isl1Cre mutant to control OFTs. Red indicates increased expression and green decreased expression in mutants. Note the reproducible direction and magnitude of the changes. In the BMP/TGFβgene list, BMP pathway members are in bold, TGFβ pathway members are in regular type and shared genes are marked with an asterisk. Fold changes are log base 2; P<0.05. (C-E) mRNA in situ hybridizations of E9.5 control and Fgf8;Isl1Cre mutants. Bmp4 expression is decreased in the OFT (arrowhead) and SHF (arrow) of (D) Fgf8;Isl1Cre and (E) Fgfr1c/c;Fgfr2c/+;Isl1Cre mutants that develop PTA. (F,G) Anti-phosphoSMAD1/5/8 immunohistochemistry on sagittal sections of control versus Fgf8;Isl1Cre mutant OFTs. Hoechst staining in blue, anti-pSMAD in red. pSMAD+ cells are abundant in control, compared with mutant, pharyngeal and subendothelial mesenchyme (arrows) and in the OFT endothelium (arrowheads). cu, proximal OFT cushion; PA, pharyngeal arch. (H-I') Anti-phosphoSMAD1/5/8 immunohistochemistry on transverse sections of control versus Spry2-GOF;Mesp1Cre mutant OFTs. Hoechst staining in blue, anti-pSMAD in red. pSMAD+ cells are abundant in control OFT endothelium (arrowheads) and in subendothelial mesenchymal cells (arrows). (H,I) Distal OFT cushions (cu). (H',I') Proximal OFT cushions. Bracket in H' shows large numbers of pSMAD+ endothelial cells in the control.

 

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