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First published online March 23, 2006
doi: 10.1242/10.1242/dev.02309

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Tbx1 affects asymmetric cardiac morphogenesis by regulating Pitx2 in the secondary heart field

¹ Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
² Ophthalmology and Visual Sciences, Cell and Developmental Biology, 350 Kellogg Eye Center University of Michigan Medical School, 1000 Wall Street, Ann Arbor, MI 48105, USA.
³ Departments of Medicine and Cell and Developmental Biology, University of Pennsylvania, 954 Biomedical Research Building (BRB) II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA.
⁴ CNR-Institute of Neurosciences, Department of Biomedical Sciences, University of Padova, Padova, Italy.

View larger version (134K): [in a new window]   Fig. 1. Co-expression of Tbx1 and Pitx2 in the SHF and asymmetric Tbx1 expression in wild-type embryos. In situ hybridization in transverse and sagittal sections through the heart shows overlapping expression of Tbx1 (A,E,I) with Pitx2 (B,F,J) within the Nkx2.5 (C,G,K) and Isl1 expression domains (D,H,L). Co-expression of Tbx1, Pitx2, Nkx2.5 and Isl1 is shown in the left pharyngeal mesoderm (pm), the left horn of the sinus venosus (lsv) and the outflow tract (oft) up to the inner curvature of the common ventricle (cv) at E8.5 (arrowheads in A-H) and E9.5 (arrowheads in I-L), respectively. (M-O) Transverse serial sections (cranial to caudal) of E9 embryos, where the asymmetric Tbx1 expression on the left side of the pharyngeal mesoderm (arrowhead in N,O) is visible. The asymmetry is limited to the most caudal part of Tbx1 expression domain. This asymmetry is retained in the Pitx2-/- mice (arrowhead in P). da, dorsal aorta; fg, foregut; lcv, left cardinal vein. Scale bars: 100 µm in A-H; 200 µm in I-L; 200 µm in M-P.

View larger version (89K): [in a new window]   Fig. 2. Downregulation of left Pitx2 expression in Tbx1-/- embryos at stages E8 and E10. (A-D) Downregulation of Pitx2 expression in the outflow tract (oft) and in the left splanchnic mesoderm (sm) is shown in sections cut from whole-mount in situ hybridized E8 Tbx1-/- embryos (B,D), compared with wild type (A,C). (E,F) Pitx2 expression levels are not changed in the most cranial part of the head mesenchyme at this stage. (G,H) Expression of Nkx2.5 in the heart is also unchanged in the Tbx1-/- embryos (H) when compared with the wild type (G). (I-N) Whole-mount in situ hybridization (I-L) and in situ hybridization in sections (M,N) show reduced Pitx2 expression in the left atrium (la), the outflow tract (oft) and right ventricle (rv) of E10.5 Tbx1-/- hearts (K,L). Pitx2 expression in wild-type hearts is shown in I and J. Absence of Pitx2 in the heart (arrows) is shown in transverse sections of E10 Tbx1-/- (N) when compared with the normal expression in Tbx1+/- (M). (O,P) The pattern and intensity of Pitx2 expression is unchanged in the same embryos in the midgut (mg) and in the umbilical vein (uv). (Q) Quantitative RT-PCR analysis of Pitx2 mRNA expression level on E10.5 whole hearts from wild type and Tbx1-/- embryos, P<0.05. The results are representative of six wild-type and 11 Tbx1-/- embryos. The error bars indicate the s.d. Gapdh expression level was used for normalization. da, dorsal aorta; hm, head mesenchyme; lv, left ventricle; ra, right atrium; nt, notochord. Scale bars: 100 µm in A-H; 300 µm in M-N.

View larger version (111K): [in a new window]   Fig. 3. Pitx2+/-; Tbx1+/- newborns and embryos exhibit cardiac defects. (A,D,E,F) Histological analysis of Pitx2+/-; Tbx1+/- newborns revealed severe heart defects such as double outlet right ventricle (dorv; arrow), stenosis of the infundibulum (arrowhead) (A), atrial septal defect (asd), ventricular septal defect (vsd) (D), atrio-ventricular valve defects (vad), a common atrio-ventricular junction (cav) (E) and abnormal drainage of pulmonary vein (pv) (F). Pitx2+/- (B,G) and Tbx1+/- (C,H) were normal. Histological analysis of Pitx2+/-; Tbx1+/- E10.5 embryos (J,L) shows grossly malformed hearts compared with wild-type embryos (I,K). ao, aorta; avc, atrio-ventricular canal; la, left atrium; lv, left ventricle; pt, pulmonary trunk; ra, right atrium; rv, right ventricle. Scale bars: 1 mm in A-H; 250 µm in I-L.

View larger version (73K): [in a new window]   Fig. 4. In situ hybridization with cardiac markers Tbx3, Tbx2, Nppa and Mhc. Sections from E10.5 wild-type (A,C,E,G) and Pitx2+/-; Tbx1+/- embryos (B,D,F,H). Staining with the Tbx3 (A,B) and Tbx2 (C,D) probes indicates correct molecular patterning in the AVC and inner curvature region. Reduced expression of the chamber marker gene Nppa is found in the Pitx2+/-; Tbx1+/- embryos (E,F), in line with the minor degree of ventricular expansion in the Pitx2+/-; Tbx1+/- embryos, as shown by marker staining for Mhc (G,H). la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle. Scale bar: 300 µm in A-H.

View larger version (36K): [in a new window]   Fig. 5. Tbx1 activates the Pitx2 enhancer (Pitx2-ASE) by binding to a T-half site within the enhancer and through interaction with Nkx2.5. (A) Luciferase assays show that Tbx1 or Nkx2.5 can activate the Pitx2-ASE reporter weakly. Co-transfection of Tbx1 and Nkx2.5 leads to a 12-fold activation of the Pitx2-ASE reporter. Data are presented as mean±s.e.m. for three independent experiments. (B) Luciferase assays using the Pitx2 enhancer mutated at the T-half site (Pitx2-Mut) do not show activation of Pitx2-Mut when transfected with either Tbx1, Nkx2.5 or both expression constructs. Data are presented as mean±s.e.m. for three independent experiments. (C) In EMSAs, Tbx1 binds to the putative T-half site found within the Pitx2 enhancer (wild type). Arrow shows wild-type Tbx1-DNA complex. Addition of Tbx1 antibody results in a supershift of the complex, marked by an arrowhead. Mutation of the T-half site (M1, M2) does not lead to the formation of a Tbx1-DNA complex. Binding of Tbx1 to the consensus T-half site (Cons) is weak. (D) Co-immunoprecipitation of co-transfected Flag-Nkx2.5 and Tbx1-GFP followed by detection of Tbx1 (arrow, upper blot) and Flag protein (arrow, bottom blot) shows interaction between Nkx2.5 and Tbx1. Untransfected cells, and cells transfected with only Tbx1 or Nkx2.5 were used as controls. Unspecific bands (upper blot) and IgG bands (bottom blot) are marked with an arrowhead.

View larger version (42K): [in a new window]   Fig. 6. Model for the regulation of cardiac morphogenesis by Tbx1 in the secondary heart field. We propose that Tbx1 exerts a dual role on the left side of the SHF. Tbx1 acts through a novel second pathway in the left cardiac precursor cells of the SHF via Pitx2. It regulates the maintenance of Pitx2c expression, originally activated in the left LPM by Nodal signaling, by interaction with Nkx2.5 to regulate cell proliferation or migration to ensure proper asymmetric cardiac morphogenesis (yellow box). This pathway complements the previously described non cell-autonomous Tbx1-Fgf8/10 pathway in the left SHF. The Tbx1-Fgf8/Fgf10 pathway is required for cell proliferation in the right and the left SHF.

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