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Regulation of left-right asymmetry by thresholds of Pitx2c activity

¹ Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA
² Department of Anatomy and Developmental Biology, St. George’s Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK

View larger version (50K): [in a new window]   Fig. 1. Gene targeting and Pitx2 isoform expression. (A,B) 8.5 dpc expression of Pitx2c (A), Pitx2a and Pitx2b (B). Arrow denotes sinous venosus and arrowhead indicates lateral mesoderm. (C) 9.0 dpc expression of Pitx2c. bw, body wall; sp, splanchnopleure. (D,E) 10.5 dpc expression of Pitx2c in wild-type (D) and ab;ab embryos (E). lb, lung bud; s, stomach. (F) X-gal staining in ab; ab guts. c, cecal diverticulum; d, duodenum; mg, midgut; sma, superior mesenteric artery. (G,H) 10.5 dpc expression of Pitx2c (G), Pitx2a and Pitx2b (H). oe, oral ectoderm; pm, periocular mesenchyme. (I,J) Eye phenotypes (arrowhead) of wild-type (I) and ab;ab (J) embryos. (K) Exon usage of Pitx2 isoforms. (L) Pitx2 genomic structure and targeting strategy. The boxes represent exons and straight lines introns. The exons are not drawn to scale. (M) Targeted allele before and after removal of the PGKneomycin cassette. At the bottom is the Pitx2-null allele that was previously generated (Lu et al., 1999). (N) Southern blot with flanking probes: tail DNA probed with the 5' flanking probe (left); tail DNA probed with the 3' flanking probe (center). The right panel shows a Southern blot probed with an internal lacZ probe after crossing the abhypoc +/- mice to the CMV cre recombinase deletor strain to generate ab +/- mice. After recombination, and PGKneomycin removal, the lacZ probe hybridizes to a 2 kb fragment, while in mice that still retain the PGKneomycin, the lacZ probe hybridizes to a 3 kb fragment. In the right-hand panel, + above the lanes denotes mice that have retained PGKneomycin and – denotes a mouse that has deleted PGKneomycin. (O) Diagram of riboprobe that distinguishes between isoforms and the Pitx2c-protected fragment. (P) Ribonuclease protection assay of mRNA from Pitx2 allelic combinations: 1, probe; 2,3, tRNA; 4,5, wild type; 6, abcnull heterozygous; 7,8, abcnull; ab; 9,10, abcnull; abhypoc; 11,12, abcnull; abcnull; 13, markers. (Q) Quantitation of Pitx2c mRNA levels in the different Pitx2 allelic combinations.

View larger version (93K): [in a new window]   Fig. 2. Cardiac phenotypes of Pitx2 allelic combinations. (A-D) Transverse sections through 11.5 dpc hearts of wild-type and ab;ab mutants. In wild-type (+;+) and ab; ab embryos, the primary interatrial septum (PIAS) extends from the spina vestibuli to the roof of the atrium (solid arrowheads, A,C) and the pulmonary vein is located at the base of the PIAS (blunt arrow in A,C). Paired venous valves are found at the boundary of the right atrium and right sinus horn (winged arrowheads in A,C). In wild-type and ab; ab hearts, the cushions of the proximal OFT occlude the lumen, and twist in characteristic fashion, with left and right lateral lumens (arrows in B,D). Moreover, the right side of the atrium has trabeculations (pectinate muscles, pm in A and C), while the left side has none. (E-H) Transverse sections through 11.5 dpc abcnull;abcnull hearts. In abcnull;abcnull mutant embryos, the PIAS is just a stub (blunt arrowhead in F) and the pulmonary vein has an anomalous connection, emptying into the right sinus horn, or saccus reuniens (arrow, E). Also in abcnull;abcnull mutant embryos, paired venous valves are present on both the left and right (winged arrowheads in F). The mutant OFT has a large lumen with symmetrical cushions (winged arrows in F-H) and the dorsal left atrium is more extensively trabeculated than the right (pm in G). The left superior caval vein, present in wild-type and ab; ab hearts (asterisks in A and C), is absent in abcnull; abcnull hearts. (I-M) Transverse sections through 12.5 dpc hearts of wild-type and abcnull;abhypoc mutants. The PIAS is normal (blunt arrowheads I and K) and the pulmonary vein is located at base of PIAS (blunt arrow K) in wild-type and abcnull;abhypoc embryos. The distal OFT is separated into the left aortic arch and the right pulmonary trunk (winged arrows in I and K) and the proximal OFT cushions form three primordia of valve leaflets in both arterial trunks, although septation is incomplete (winged arrows, J,M). The right atrium in wild-type hearts has trabeculations (pm, I), while the left atrium has none. The dorsal left and right atria of abcnull; abhypoc hearts have similar degrees of trabeculation (pm, L). The superior and inferior AV cushions have fused in wild-type and abcnull; abhypoc hearts (asterisks, J,M), forming separate left and right AV canals. (N-P) Transverse sections through 12.5 dpc abcnull;abcnull hearts. In abcnull;abcnull mutants, the PIAS is truncated (blunt arrowheads, O) and the pulmonary vein is found within dorsal mesocardium, but caudal to the atrial wall, so emptying into the right sinus horn, or saccus reuniens (blunt arrow, N). The pulmonary vein has exits to both the left side (blunt arrow, O) and the right side (not shown) in abcnull;abcnull embryos. The abcnull;abcnull mutant distal OFT is unseptated, with malaligned arterial trunks (winged arrow, N) and the OFT cushions are malaligned (winged arrow, P). Moreover, the AV cushions have not fused, and there is a common AV junction (asterisk, P) in abcnull;abcnull embryos. The left superior caval vein shown in wild-type and abcnull; abhypoc hearts (asterisks in I,K,L) is absent in abcnull; abcnull hearts.

View larger version (106K): [in a new window]   Fig. 3. Lung phenotypes of Pitx2 allelic combinations. (A,B) Diagram of asymmetric lung branching pattern. (C,D) Whole-mount in situ hybridization with the sonic hedgehog probe in 9.0 dpc wild-type (C) and abcnull; abcnull (D) embryos. (E-I) Dorsal aspect of adult wild-type (E), neonatal abcnull; abhypoc (F), abcnull; ab (G), abcnull +/- (H) or oblique view of ab; ab (I). Ca, caudal lobe; Crl, cranial lobe; LL, left lung. (J-N) 11.5 dpc whole-mount in situ with probe for sonic hedgehog: wild-type (J), abcnull; abcnull (K), abcnull; abhypoc (L), abcnull; ab (M) and ab; ab (N). (O-R) 12.0 dpc whole-mount in situ with probe for bone morphogenetic protein 4: wild-type (O), abcnull; abcnull (P), abcnull; abhypoc (Q) and abcnull; ab (R).

View larger version (88K): [in a new window]   Fig. 4. Gut phenotypes of Pitx2 allelic combinations. (A-E) Gut looping in 10.5 dpc embryos visualized using the probe for sonic hedgehog. Genotypes are shown. (F-J) Duodenal rotation in 12.5 dpc embryos visualized using the probe for sonic hedgehog. Genotypes are shown. Direction of duodenal rotation is outlined. (K-M) Pitx2c expression in duodenum shown by whole-mount in situ hybridization. Note bilateral expression as denoted by the arrows. (N,O) Normal and arrested midgut rotation in wild-type (N) and ab;ab mutant (O) embryos. Direction of midgut looping is outlined. Annular pancreas in ab;ab mutant embryo (O) is denoted by arrows.

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