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Fig. 3. Cited2-null embryos have abnormal expression of left sided determinants. RNA in situ hybridization of embryos and hearts hybridized with probes that recognize Nodal (A-C), both Lefty1 and Lefty2 (D-I), and Pitx2 (J-R) transcripts. Nodal is expressed in the left lateral mesoderm (arrow) and node of six- to seven-somite stage Cited2 wild-type (A) and heterozygous (B), but not in LPM of Cited2-null (C) embryos. (D-I) A combined Lefty1/2 probe shows that Lefty1 and Lefty2 are expressed in the left prospective floor plate of the neural tube (white arrow), and left LPM (black arrow) respectively, in seven- to nine-somite stage Cited2-wild-type (D,G) and heterozygous (E,H) embryos. In Cited2-null embryos (F,I), Lefty2 expression is not detected, whereas the expression of Lefty1 is restricted to the posterior floor plate. (J-L) Pitx2 is expressed in left lateral mesoderm (arrow) of 12- to 14-somite stage Cited2 wild-type (J) and heterozygous (K), but not Cited2-null (L) embryos. (M-R) At 13.5 dpc, Pitx2 is expressed in the ventral myocardium of the right ventricle, throughout the left atrium and in ventrally viewed veins of Cited2 wild-type embryos (M,P) and in null embryos that lack a laterality defect (N,Q). Pitx2 is not expressed in the hearts of Cited2-null embryos that have a right isomeric phenotype (O,R). In some Cited2-null hearts, a novel domain of Pitx2 expression was observed in dorsal ventricular cells (Q; arrow). Ventral view (A-C,J-O), lateral view (D-F), posterior view (G-I), dorsal view (P-R). Scale bar: 235 µm in A-C; 175 µm in D-I; 365 µm in J-L; 335 µm in M-R.

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