Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium

Abstract

Current models of left-right asymmetry hold that an early asymmetric signal is generated at the node and transduced to lateral plate mesoderm in a linear signal transduction cascade through the function of the Nodal signaling molecule. The Pitx2 homeobox gene functions at the final stages of this cascade to direct asymmetric morphogenesis of selected organs including the heart. We previously showed that Pitx2 regulated an asymmetric pathway that was independent of cardiac looping suggesting a second asymmetric cardiac pathway. It has been proposed that in the cardiac outflow tract Pitx2 functions in both cardiac neural crest, as a target of canonical Wnt-signaling, and in the mesoderm-derived cardiac second lineage. We used fate mapping, conditional loss of function, and chimera analysis in mice to investigate the role of Pitx2 in outflow tract morphogenesis. Our findings reveal that Pitx2 is dispensable in the cardiac neural crest but functions in second lineage myocardium revealing that this cardiac progenitor field is patterned asymmetrically.

Fig. 1

CNC marker analysis in Pitx2^null mutant embryos. In situ hybridization analysis with Ap2 probe (A, B) and Crabp probe (C, D) on 9.5-dpc wild-type and Pitx2^null mutant embryos. Black arrows denote CNCs expression in branchial arch mesoderm and no obvious change between wild-type and Pitx2 mutant. (E–H) Left side view of whole-mount lacZ staining (E, F) and sagittal sections (G, H) of Wnt-1^cre;R26R on 9.5-dpc wild-type (E, G) and 9.5-dpc Pitx2^null mutant embryos (F, H) indicating that CNC’s migrate into the 3rd and 4th branchial arches (arrows denoted). Arrowheads denote the CNCs in the OFT. (I, J) Frontal view of whole-mount lacZ staining of Wnt-1^cre;R26R on 9.5-dpc wild-type (I) and Pitx2^null mutant (J) to show CNC’s migrate into outflow tract (arrows). TopGal activity in 12.5-dpc wild-type embryo (K) and Pitx2^null mutant (L). Arrows denote TopGal Activity. OFT, outflow tract; la, left atrium; lv, left ventricle; rv, right ventricle.

Fig. 2

Conditional inactivation of Pitx2 in the CNC. Serial coronal sections show systemic circulation of 14.5-dpc wild-type (A) and Wnt1^Cre;Pitx2 (n/f) heart (C, E, G). Arrows denote that the aortas connect to the left ventricle. Serial coronal sections show pulmonic circulation of 14.5-dpc wild-type (B) and Wnt1^Cre;Pitx2 (n/f) heart (D, F, H). Arrows denote the right connection between pulmonary arteries and right ventricles. la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle.

Fig. 3

Pitx2^cre lineage analysis and Pitx2^creERT2 fate mapping. (A) Summary of exon usage by Pitx2 isoforms. (B, C) Frontal view of LacZ staining of a dissected 11.5-dpc (B) and 14.5 dpc (C) Pitx2^cre;R26R hearts and a transverse section through the 11.5 dpc OFT (D). Pitx2 daughters denoted by an arrow (OFT myocardium) and arrowheads (endocardium). (E) Pitx2 genomic structure, Pitx2^creERT2 knock-in targeting strategy, and targeted clones confirmed by southern blot. Boxes represent exons and straight lines, introns. P1 and P2 indicate two promoters that initiate transcription of different isoforms. (F–H) LacZ staining of 12.5-dpc Pitx2 ^creERT2; R26R hearts, labeled at 9.5 dpc and 10.5 dpc. (I) Transverse section of OFT in panel G. Arrows denote Pitx2 daughter cells. la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle.

Fig. 4

Conditional inactivation of Pitx2 in the SHF. Dye injection into 14.5-dpc hearts to show OFT morphology with schematic diagram below. (A, E) Wild-type, (B, F) Mef2c^cre;Pitx2^n/f showing TGA, (C, G) Isl1^cre;Pitx2^n/f showing DORV, (D, H) Pitx2^null mutant showing TGA. Note that each diagram is associated with its above picture. Arrows denote vessel alignment. TGA, transposition of great artery; DORV, double outlet of right ventricle; ao, aorta; pt, pulmonary trunk; la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle.

Fig. 5

Sections of Pitx2 SHF deficient embryos. Sagittal sections through 13.5-dpc (A–C), and 14.5-dpc (D–F) embryos. (C, F) Higher magnification views of boxed areas in panels B and E. (G–I) Modified transverse sections of 14.5-dpc Mef2c^cre; Pitx2^n/f heart showing abnormal arterioventricular alignment. (I) Higher magnification image of boxed area in panel H. Genotypes and stages are labeled. ao, aorta; pt, pulmonary trunk; la, left atrium; ra, right atrium.

Fig. 6

Secondary heart field marker analysis in Pitx2 null mutants. Right lateral view of Isl1 fate mapping in 9.5 dpc control (A) and Pitx2^null mutant embryo (B). Arrow and arrowhead denote distal and proximal OFT respectively. Circled area in panel B denotes the area of reduced LacZ staining. (C, D) Left views of whole-mount in situ hybridization with Isl1 probe in 9.5 dpc control (C) and Pitx2 mutant embryo (D). Arrows denote branchial arch mesoderm. (E) Bar graph quantitation of OFT size based on morphometric analysis as described in Materials and methods. (*P < 0.02, Student’s t-test). (F, G) PCNA assay shows that cell proliferation is reduced in Pitx2 mutant proximal OFT compared to control. Arrowheads denote proximal outflow tract. (H) Bar graph of proliferation Index is shown in panel H. (*P < 0.01, Student’s t-test). ba1, first branchial arch; OFT, outflow tract; AVC, atrioventricular canal.

Fig. 7

Chimera analysis of Pitx2 function in the OFT. (A, B) Lineage tracing with the Pitx2^cre and R26R alleles in control (A, C, E) and Pitx2 mutant embryos (B, D, F). Pitx2 daughter cells are stained with LacZ. Arrows denote the Pitx2 daughters that are normally contribute to proximal aortic myocardium and are deficient in the mutant embryos. Note that the Pitx2c allele is a deletion of the Pitx2c isoform that allows for analysis of later stage embryos. (G–I) Pitx2+/− ↔wild type chimeras at 13.5 dpc. Percentage chimerism is labeled and Pitx2 daughters in the OFT denoted by arrowhead and in RV by the arrows. (J–M) Pitx2−/− ↔wild type chimeras at 13.5 pc. Percentage chimerism is shown. Pitx2 daughters in the OFT denoted by arrowhead and in RV by the arrows. Angled arrows indicate the Pitx2 mutant daughters that abnormally persist at the junction of the aorta and pulmonary trunk. (N, O) Sections through the OFT of Pitx2+/− ↔ wild type chimeras (N) and Pitx2−/− ↔ wild type chimera (O). Arrows indicate cells that abnormally persist in the mutant chimera. ao, aorta; pt, pulmonary trunk; la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle.