Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling

Abstract

Heparin-binding epidermal growth factor (HB-EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB-EGF and BTC alleles by homologous recombination. Most HB-EGF(-/-) mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC(-/-) mice were viable and fertile and displayed no overt defects, the lifespan of double null HB-EGF(-/-)/BTC(-/-) mice was further reduced, apparently due to accelerated heart failure. HB-EGF(-/-) newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB-EGF transcripts were localized to endocardial cells lining the margins of wild-type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor-alpha converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE-derived soluble HB-EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context.

Fig. 1. Targeted disruption of HB-EGF and BTC. (A) Schematics illustrate strategies used to inactivate the mouse HB-EGF and BTC alleles. Targeting constructs were designed to eliminate exons (filled boxes) 1 and 2 of the HB-EGF gene, or exon 3 of the BTC gene using genomic sequences isolated from a mouse 129/Sv library. Heavy lines denote 5′ and 3′ arms of homology, and dashed lines bracket the genomic regions replaced by the Neo cassette. Hatched boxes mark probes used to verify correct targeting, and the locations of useful restriction sites and resulting fragments are noted. B, BglII; E, EcoRI; H, HindIII; N, NcoI; Nt, NotI; S, StuI. (B) Southern blot analyses of genomic DNAs digested with NcoI, showing results diagnostic for wild-type, heterozygous and homozygous targeted alleles using the indicated probes. (C) Genomic PCR using exon- or Neo-specific primers. (D) Northern blots of lung RNA from mice of the designated genotypes. Arrows indicate the apparent mass of DNA or RNA species. Ntds, nucleotides; kb, kilobases.

Fig. 1. Targeted disruption of HB-EGF and BTC. (A) Schematics illustrate strategies used to inactivate the mouse HB-EGF and BTC alleles. Targeting constructs were designed to eliminate exons (filled boxes) 1 and 2 of the HB-EGF gene, or exon 3 of the BTC gene using genomic sequences isolated from a mouse 129/Sv library. Heavy lines denote 5′ and 3′ arms of homology, and dashed lines bracket the genomic regions replaced by the Neo cassette. Hatched boxes mark probes used to verify correct targeting, and the locations of useful restriction sites and resulting fragments are noted. B, BglII; E, EcoRI; H, HindIII; N, NcoI; Nt, NotI; S, StuI. (B) Southern blot analyses of genomic DNAs digested with NcoI, showing results diagnostic for wild-type, heterozygous and homozygous targeted alleles using the indicated probes. (C) Genomic PCR using exon- or Neo-specific primers. (D) Northern blots of lung RNA from mice of the designated genotypes. Arrows indicate the apparent mass of DNA or RNA species. Ntds, nucleotides; kb, kilobases.

Fig. 2. Similarly defective cardiac valves in HB-EGF^–/–, EGFR^–/– and TACE^–/– mice. (A–L) H&E sections of newborn (P1) hearts of the indicated genotypes showing atrioventricular (AV) and semilunar valves (arrows). Scale bar = 500 µm. (M) Measurement of aortic valve areas (mm²) ± SD of the indicated P1 mutant hearts, including those of rescued ErbB4^–/– mice, with their respective +/+ controls. Asterisks denote statistically significant results.

Fig. 2. Similarly defective cardiac valves in HB-EGF^–/–, EGFR^–/– and TACE^–/– mice. (A–L) H&E sections of newborn (P1) hearts of the indicated genotypes showing atrioventricular (AV) and semilunar valves (arrows). Scale bar = 500 µm. (M) Measurement of aortic valve areas (mm²) ± SD of the indicated P1 mutant hearts, including those of rescued ErbB4^–/– mice, with their respective +/+ controls. Asterisks denote statistically significant results.

Fig. 3. In situ detection of HB-EGF, EGFR, ErbB3, ErbB4 and TACE transcripts in wild-type cardiac valves. (A–F) HB-EGF; (G and H) EGFR; (I and J) ErbB3; (K and L) ErbB4; (M and N) TACE. Arrows mark regions of high expression. Sense riboprobes were hybridized to confirm specific hybridization in each case (data not shown). AoV, aortic valve; bf, bright field; df, dark field; v, valves. Scale bars = 100 µm.

Fig. 3. In situ detection of HB-EGF, EGFR, ErbB3, ErbB4 and TACE transcripts in wild-type cardiac valves. (A–F) HB-EGF; (G and H) EGFR; (I and J) ErbB3; (K and L) ErbB4; (M and N) TACE. Arrows mark regions of high expression. Sense riboprobes were hybridized to confirm specific hybridization in each case (data not shown). AoV, aortic valve; bf, bright field; df, dark field; v, valves. Scale bars = 100 µm.

Fig. 4. Defective remodeling of cardiac valves in mutant mouse hearts. (A–H) H&E sections of HB-EGF^+/+ or HB-EGF^–/– hearts showing (A–D) atrioventricular (AV) valves or (E–H) pulmonary valves (PV) at the indicated time points. (I–P) H&E sections of heart showing AV valves or PV valves of the indicated TACE or EGFR genotypes at E15.5. Arrows denote valves. Scale bars = 100 µm. (Q–T) Quantitative comparison of valvulogenesis in HB-EGF^+/+ or HB-EGF^–/– hearts. (Q and R) Cushion/heart ratio ± SD at the indicated ages. (S) Percentage of TUNEL-positive cells at the indicated ages in AV valves or PV; (T) percentage of BrdU-positive cells. Filled bars, HB-EGF^+/+; unfilled bars, HB-EGF^–/–. Asterisks denote statistically significant results.

Fig. 4. Defective remodeling of cardiac valves in mutant mouse hearts. (A–H) H&E sections of HB-EGF^+/+ or HB-EGF^–/– hearts showing (A–D) atrioventricular (AV) valves or (E–H) pulmonary valves (PV) at the indicated time points. (I–P) H&E sections of heart showing AV valves or PV valves of the indicated TACE or EGFR genotypes at E15.5. Arrows denote valves. Scale bars = 100 µm. (Q–T) Quantitative comparison of valvulogenesis in HB-EGF^+/+ or HB-EGF^–/– hearts. (Q and R) Cushion/heart ratio ± SD at the indicated ages. (S) Percentage of TUNEL-positive cells at the indicated ages in AV valves or PV; (T) percentage of BrdU-positive cells. Filled bars, HB-EGF^+/+; unfilled bars, HB-EGF^–/–. Asterisks denote statistically significant results.

Fig. 5. Disregulated Smad signaling in HB-EGF mutant cushions. Sections of (A and B) pulmonary valve (PV) or (C and D) aortic valve (AoV) from E14.5 immunostained with phospho-Smad1/5/8 antibody and counterstained with hematoxylin. Arrows mark positive staining cells. Scale bar = 100 µM.

Fig. 6. Cardiac defects in HB-EGF^–/– and HB-EGF^–/–/BTC^–/– mice. (A) Hearts from 6-week-old male mice. (B and C) H&E-stained cross- sections of 6-week-old (B) HB-EGF^+/+ and (C) HB-EGF^–/– hearts. (D and E) PAS-stained transverse sections of left ventricle cardiomyocytes. (F) PAS-stained section of thickened aortic valve leaflet from 6-month-old HB-EGF^–/– heart. White arrow marks chondrocytes; black arrows denote ectopic cartilage formation. (G) H&E-stained section of HB-EGF^–/–/BTC^–/– heart. Arrow marks a thrombus in the left atrium. (H and I) Masson trichrome-stained sections of HB-EGF^–/–/BTC^–/– hearts. Arrows mark extensive intraventricular (H) or focal epicardial (I) fibrosis (blue stain) within or on the left ventricle. v, ventricle; ao, aorta; lv, left ventricle. Scale bars = 3.5 mm (A), 1 mm (B and C), 20 µm (D and E), 100 µm (F), 1 mm (G–I).

Fig. 7. A comparison of lung morphology and maturity in HB-EGF^+/+ and HB-EGF^–/– mice. (A and B) H&E-stained sections of newborn lungs. Arrows point to thickened mesenchyme. (C and D) PAS-stained sections of newborn lung. Arrows (D) point to areas enriched in cells with high glycogen content. (E and F) Sections of newborn lung immunostained for prosurfactant C. Arrows point to positive cells. (G) Bars indicate mean number of alveoli counted from duplicate histological sections of newborn lung (n = 6–9 mice) of the indicated genotypes ± SD. Asterisk denotes significant result (P = 0.0012). Scale bars = 200 µm (A and B), 100 µm (C–F).