Pitx2-mediated cardiac outflow tract remodeling

Abstract

Background: Heart morphogenesis involves sequential anatomical changes from a linear tube of a single channel peristaltic pump to a four-chamber structure with two channels controlled by one-way valves. The developing heart undergoes continuous remodeling, including septation.

Results: Pitx2-null mice are characterized by cardiac septational defects of the atria, ventricles, and outflow tract. Pitx2-null mice also exhibited a short outflow tract, including unseptated conus and deformed endocardial cushions. Cushions were characterized with a jelly-like structure, rather than the distinct membrane-looking leaflets, indicating that endothelial mesenchymal transition was impaired in Pitx2(-/-) embryos. Mesoderm cells from the branchial arches and neural crest cells from the otic region contribute to the development of the endocardial cushions, and both were reduced in number. Members of the Fgf and Bmp families exhibited altered expression levels in the mutants.

Conclusions: We suggest that Pitx2 is involved in the cardiac outflow tract septation by promoting and/or maintaining the number and the remodeling process of the mesoderm progenitor cells. Pitx2 influences the expression of transcription factors and signaling molecules involved in the differentiation of the cushion mesenchyme during heart development.

Figure 1. Shorter and Hypocellular Cardiac OT in Pitx2 Mutants

Ventral view of the entire heart at E10.5 (A, B) and E12.5 (E, F) showed a shortened OT with a prominent DORV in Pitx2-mutant mice (F). (C, G) The length of the OT was measured as indicated by brackets. Statistics were based on results from 3 different embryos at each stage. HE staining on 14 µm transverse cryosections at E10.5 (A1, B1) and E 12.5 (E1, F1) mice indicated thinner OT epithelium in the conus. The black and yellow lines correspond to the outer and inner epithelium, respectively. (D, H) Cell counts of a set of 5–8 serial sections along the OT showed reduction of cells in the cushions of mutants. ***: p<0.01, **: p<0.05. (I, J) Double labeling immunohistochemistry on E12.5 mouse transverse sections for MF20 and Pitx2 (β-Gal). No MF20⁺ cells were detected in the conal septum and semilunar valves in the mutants. Ao, aorta; AV, aortic valve; LV, left ventricle; PA, pulmonary artery; PV, pulmonary valve; RV, right ventricle.

Figure 2. OT Cushion Mesenchymal Cell Proliferation and Apoptosis Defects in Pitx2 Mutants

TUNEL assay (A–D) was performed on 14 µm frontal sections to identify the cell apoptosis index during OT remodeling. TUNEL signal was not detected at E10.5 in either OT mesenchyme of the heterozygote or in mutant littermates (A, B). The TUNEL signal was detected in heterozygote OT cushion mesenchyme (C, white arrows) but not in mutant littermate (D) at E12.5. The number of apoptotic cells in the OT cushion was counted based on eight continuous sections for three individual embryos at E12.5 (E). Double labeling of β-gal and Bromodeoxyuridine (BrdU) on 14 µm frontal sections showed Pitx2 effects on cell proliferation (F, G, I, J). The number of proliferative cells was increased in Pitx2 mutants at E10.5 and E12.5, respectively (H, K). The BrdU⁺ and BrdU⁺/Pitx2⁺ cells were counted based on five continuous sections for three individual embryos in each stage. The OT cushion was traced by a white line. Statistics were based on results from 3 different embryos at each stage. ***: p<0.01.

Figure 3. Defects of the Second Cardiac Cell Lineage in Pitx2 Mutants

Double labeling immunohistochemistry of transverse cryosections sections of Mef2c^Cre/+|Rosa^EGFP/+|Pitx2^+/+ (A, D, G) and Mef2c^Cre/+|Rosa^EGFP/+|Pitx2^Z/Z (B, E, H) for EGFP (Mef2c) and Isl1 indicated reduction of cell populations in the mutant BA (B) at E10.5 and OT at E10.5 (E) and E12.5 (H). (G, F, I) Quantitative analysis by qPCR for EGFP and Isl1 indicated reduced levels in E10.5 BA (C) and OT (F, I). ***: p<0.01; **: p<0.05; *: p<0.1.

Figure 4. Impaired cNC cells in Pitx2 mutants

Wnt1^Cre/+|Rosa^EGFP|Pitx2^+/+ (A, C, E) and Wnt1^Cre/+|Rosa^EGFP|Pitx2^Z/Z (B, D, F) hearts were dissected at E 9.5 (A, B), E 10.5 (C, D) and E 12.5 (E, F). The green fluorescent cNC cells that migrated towards the OT were reduced in mutants, with more prominent phenotype at E9.5 and E10.5. (G, H) Whole mount RNA in situ hybridization at E12.5 hearts for Ap2α expression indicated reduced levels in the great arteries in mutants.

Figure 5. Pitx2 Occupancy on SSTF Gene Loci in BA and Heart Chromatin

Sonically sheared chromatin, isolated from E10.5 mice, was used to detect Pitx2 protein occupancy on Mef2c (A) and Isl1 (B) in BA biopsies and Gata4 (E) and Nkx2.5 (G) in heart biopsies. PCR amplicons of 70–150bp (red boxes) were designed around highly evolutionarily conserved bicoid core motif TAATCY. Each red diamond indicated a single vertebrate species, containing the biocoid core motif. Bar graphs show the average relative amount of signal precipitated from wild type (white bar) and mutant (black bar). Pitx2 was found to occupy the Mef2c and Isl1 gene on the conserved −521 (B) and +1983 (D) sites in E10.5 embryonic BA biopsies, respectively. No significant difference was measured for Pitx2 occupancy in conserved regions of Gata4 (F) and Nkx2.5 (H) in E10.5 heart biopsies.

Figure 6. Alteration of FGF, BMP and Notch Signaling in Pitx2 Mutants

RNA in situ hybridization on cryostat sections revealed altered Fgf8 (A–C), Fgf3 (E–G), Notch2 (I–K) and Bmp4 (M–O) expression in E10.5 Pitx2 control (wild type and heterozygote) and mutant mice. The Fgf8 expression in 3^rd and 4^th BA ectoderm was decreased in mutants compared to control embryos (C). Fgf3 expression levels were reduced in the 2^nd – 6^th BA mutants (G). Notch2 expression levels, detected in the 3^rd and 4^th BA in wild type (I) and heterozygote (J), were barely detectable in 3^rd and 4^th BA in mutants (K). No Bmp4 expression was detected in the OT mesenchymal cushions (area inside the black dotted line) in mutants (O) compare to the control (M, N). Thinner OT wall (between red and black dotted line) was consistently observed in Pitx2 mutants (O). Quantitative PCR assay indicated the significantly decreased mRNA expression levels of Fgf8 (D), Fgf3 (H), Notch2 (L) and Bmp4 (P) at E10.5 BA (D, H, L) and heart (P) biopsies, respectively.

Figure 7. Vascular and Nervous System Defects in Pitx2 Mutants

Whole mount antibody staining with PECAM (A, B, D, E) and NF (G, H, J, K) was performed on E10.5 Pitx2 wild type and mutant mice. PECAM expression is reduced in BA (3, 4 and 6) and OT (A, B, arrowhead). The dorsal view of the OT (D, E) indicated the septational vasculature (arrow) was disrupted in Pitx2-null mice. (C, F) Quantitative qPCR for PECAM mRNA levels in Pitx2 wild type, heterozygote and mutant in BAs (C) and heart (F) biopsies. The expression levels of PECAM in heart were significantly decreased in E10.5 mutants. Innervation of BAs and OT was detected by NF antibody in both control and mutant embryos (G, H, J, K). V2 (red dotted line) and V3 (stars) and X (arrow head) were shorter and thinner in mutants (H). NF signals were reduced in mutant hearts (K). Quantitative qPCR assay indicated significantly decreased levels in mutants in BA (I) and OT (L) biopsies, with no difference between wild type and heterozygote at this stage. V2: maxillary nerve; V3: Mandibular nerve; VII/VIII: Facial nerve; IX: Glossopharyngeal nerve; X: Vagus nerve.