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First published online 2 October 2008
doi: 10.1242/dev.025361

Development 135, 3611-3622 (2008)
Published by The Company of Biologists 2008
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Frs2{alpha}-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

Jue Zhang1, Yongshun Lin1, Yongyou Zhang1, Yongsheng Lan1, Chunhong Lin1, Anne M. Moon2, Robert J. Schwartz3, James F. Martin1 and Fen Wang1,*

1 Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030, USA.
2 Department of Pediatrics and Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.
3 Center for Molecular Development and Disease, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Diminished Frs2{alpha} expression in Frs2{alpha}cn outflow tract and second heart field. Expression of Frs2{alpha} in (A) 0-somite stage (ss) or (B) E9.5 mouse embryos was assessed by immunostaining with anti-FRS2{alpha} antibody (green) on paraffin sections. Nuclei were stained with To-Pro3 (a DNA stain, red). a1-3, b1-3 and c1-3 are high-magnification views of the boxed areas in a, b and c, respectively. Note that in Frs2{alpha}cn/Nkx mutants, Frs2{alpha} expression was diminished in ventricular myocardium (V-M), outflow tract myocardium (OFT-M), endocardium (OFT-E), pharyngeal endoderm (PE) and splanchnic mesoderm (SM). In Frs2{alpha}cn/Mef mutants, Frs2{alpha} expression was disrupted in V-M, OFT-M and SM, but was intact in the OFT-E and PE. f, floxed allele.

 

Figure 2
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  		Fig. 2. OFT alignment and septation defects in Frs2{alpha}cn mutants. H&E staining of E14.5 mouse embryonic heart sections demonstrates overriding aorta (A-C) and double-outlet right ventricle (D-F) defects in Frs2{alpha}cn/Nkx and Frs2{alpha}cn/Mef mutants, and persistent truncus arteriosus defects in the Frs2{alpha}cn/Nkx mutant (G-I). Ao, aorta; DORV, double-outlet right ventricle; LV, left ventricle; OA, overriding aorta; RV, right ventricle; PT, pulmonary trunk; PTA, persistent truncus arteriosus. Black arrows denote OA-associated ventricular septal defects; red arrow denotes the PTA; arrowheads denote DORV.

 

Figure 3
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  		Fig. 3. Ablation of Frs2{alpha} compromises expansion of SHF progenitor cells to the OFT myocardium. (A) Sections from Frs2{alpha}f/f (a) Frs2{alpha}cn/Nkx (b) and Frs2{alpha}cn/Mef (c) mouse embryos immunostained with anti-ISL1 (green) and anti-phosphorylated histone H3 (red) antibodies. Nuclei were counterstained with To-Pro3 (blue). The total and proliferating ISL1+ cell numbers in the OFT and SM from four individuals are shown in d,e (mean ±s.d.). (B) Reduced OFT length and SHF lineage in Frs2{alpha} mutants (a-c). The OFT lengths at E9.5 as measured from six individuals are presented in d (mean ±s.d.). (e,f) X-Gal staining. The blue staining represents cells from SHF progenitors in which the R26R reporter was activated. The outline of the X-Gal-stained area in the control (e) has been superimposed on the mutant (f) to better illustrate the difference. Insets are sections from the same tissues demonstrating OFT myocardium derived from the Mef2cCre lineage (black arrows). (C) Compromised ERK1/2, but not AKT, phosphorylation in the Frs2{alpha}cn/Nkx OFT and SM. Embryo sections were immunostained with anti-phosphorylated ERK1/2 (a-c) or phosphorylated AKT (d-f) antibodies (green). Nuclei were counterstained with To-Pro3 (red). (D) Inhibition of the MAP kinase, but not the PI3K/AKT, pathway reduces the contribution of ISL1+ cells to the OFT. (a-c) Short-term (24 hour) cultures of E8.5 embryos were sectioned and stained by anti-ISL1 antibody (green) and To-Pro3 (red). Insets are high-magnification views of the same sections. (d) Cultured E8.5 embryos were treated with ERK1/2 or PI3K inhibitors as indicated. Adjacent sections from the same embryos were immunostained with anti-phosphorylated ERK1/2 or AKT (green) and with To-Pro3 (red), demonstrating the specificity and efficacy of the ERK1/2 and PI3K inhibitors. w, wild-type allele.

 

Figure 4
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  		Fig. 4. Ablation of Frs2{alpha} disrupts OFT cushion formation by inhibiting endocardial EMT and NCC contribution. (A,B) Reduced cellularity in Frs2{alpha} mutant OFT cushions. (A) Sagittal sections of E10.5 mouse embryos were H&E stained. High-magnification views of boxed areas representing proximal and distal OFT are shown as indicated (a1-c2). (B) Cell numbers in the proximal and distal cushions (Cu) were assessed and statistical data from four individuals are presented (mean ±s.d.). Note that Frs2{alpha}cn/Nkx, but not Frs2{alpha}cn/Mef, OFT cushions had decreased cellularity. En, endocardium. (C) H&E staining of transverse sections of E11.5 distal OFT showing the fusion defect in Frs2{alpha}cn/Nkx (b) OFT cushions. (D) Immunostaining for PECAM (a-c) and NFATC1 (d-f) shows compromised EMT in the Frs2{alpha}cn/Nkx proximal OFT cushions. Note that both PECAM and NFATC1 are still expressed in Frs2{alpha}cn/Nkx cushion cells (arrows). (E) Ex vivo culture of E9.5 OFTs shows compromised EMT in Frs2{alpha}cn (b) OFT myocardium. (F) Increased immunostaining for VEGFA in the Frs2{alpha}cn/Nkx OFT myocardium (My). Expression of VEGFA in E9.5 (a-c) and E10.5 (d-f) embryos. Nuclei were stained with To-Pro3. Boxed insets are high-magnification views of the myocardium. Note that VEGF expression in Frs2{alpha}cn/Nkx myocardial cells is significantly increased. (G) FRS2{alpha} is essential for FGF2 to activate NFAT transcriptional activity. Mouse embryonic fibroblasts carrying homozygous Frs2{alpha}flox alleles were transfected with an NFAT-dependent luciferase reporter with or without Cre coexpression. The cells were cultured in the presence or absence of 2 ng/ml FGF2 as indicated. Luciferase activity was then assessed. Data are mean ±s.d. of triplicate samples.

 

Figure 5
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  		Fig. 5. Compromised NCC contribution to the Frs2{alpha}cn/Nkx OFT cushions. (A) Immunostaining with anti-AP2{alpha} antibody reveals reduced numbers of migrating NCCs in the aortic sac (AS) and in pharyngeal arches (PAs) 3 and 4/6 in Frs2{alpha}cn/Nkx, but not in Frs2{alpha}cn/Mef, mouse embryos at E9.5. (B) The numbers of AP2{alpha}+ cells in the aortic sac and in pharyngeal arches 3 and 4/6 as scored from five individuals (mean ±s.d.). (C) Co-immunostaining reveals that proliferation of cardiac NCCs is compromised in Frs2{alpha}cn/Nkx embryos. Proliferating cells are labeled with anti-phosphorylated histone H3 antibody (red), NCCs with anti-AP2{alpha} antibody (green) and nuclei with To-Pro3 (blue). Triple-positive cells are indicated by arrows. (D) Statistical analyses of proliferating cardiac NCCs from five individuals (mean ±s.d.).

 

Figure 6
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  		Fig. 6. Compromised BMP4 signaling in Frs2{alpha} mutant embryos. (A) Whole-mount in situ hybridization with antisense Bmp4 probe on E9.5 (a-c) or E10.5 (d-f) mouse embryos. Sections (g-l) confirm the decreased Bmp4 expression in the Frs2{alpha}cn OFT and pharyngeal arches. (B) Phosphorylated SMAD1/5/8 expression assessed in E9.5 embryos. High-magnification views of boxed areas in b,d are shown in b',b'',d',d'',as indicated.

 

Figure 7
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  		Fig. 7. OFT defects in Fgfr1 and Fgfr2 double conditional-null embryos. (A) Whole-mount in situ hybridization demonstrating Fgfr1 (a) and Fgfr2 (d) expression in E9.5 mouse embryos. Cryosections reveal detailed expression patterns of Fgfr1 (b,c) and Fgfr2 (e,f). b1 and e1 are higher magnification views of b and e, respectively. (B) H&E staining of E14.5 embryo sections demonstrates that both Fgfr2 conditional mutant and Fgfr1/Fgfr2 (Fgfr1/r2) double conditional mutants have ventricular septal defects (VSDs) (g,h) and DROV (c,d,h), that Fgfr1/Fgfr2 have PTA (l), and Fgfr1 mutants have normal OFTs (b,f,j). Black arrowheads denote OA-associated VSDs, white arrowheads denote DROV, and the red arrow denotes PTA.

 

Figure 8
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  		Fig. 8. Double ablation of Fgfr1/Fgfr2 disrupts OFT cushion formation. (A) H&E staining of sagittal sections of E10.5 mouse OFTs. High-magnification views of the boxed areas in a,b representing proximal (p) and distal (d) OFT are shown in a1,a2,b1,b2. Note the reduced cellularity in both distal and proximal parts of mutant OFT cushions. (B) Compromised EMT in the Fgfr1/Fgfr2 double ablation (Fgfr1/r2cn/Nkx) OFT endocardium. Immunostaining reveals sustained PECAM expression in mutant OFT cushion cells (a,b) and increased VEGFA expression in the mutant OFT myocardium (c,d). Insets are high-magnification views of the same section. (C) Reduced proliferation of cardiac NCCs upon Fgfr1/Fgfr2 double ablation. (a-d) Proliferating cells were labeled with anti-phosphorylated histone 3 (pH3, red), NCCs with anti-AP2{alpha}+ antibody (green), and nuclei with To-Pro3 (blue). Triple-positive cells are indicated by arrows. (e,f) The number of AP2{alpha}+ and triple-positive cells from four individuals (mean ±s.d.). (D) (a-f) Embryos at the 4 ss stained with the indicated antibodies and To-Pro3 demonstrate that total and proliferating ISL1+ cells in the SHF are reduced in the mutants. Note that only MAP kinase, but not AKT, phosphorylation is compromised in the mutants. Yellow arrows indicate proliferating ISL1+ cells. (g,h) Total ISL1+ and proliferating ISL1+ cell numbers in the OFT and SM (mean ±s.d.). F/F, Fgfr1/Fgfr2 double-floxed embryos; CN, Fgfr1/Fgfr2 double conditional-null mutants.

 

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© The Company of Biologists Ltd 2008