Fig. 6. Loss of Lefty gene expression and delayed activation of Pitx2 activation in Nodal^600/600 embryos. Whole-mount in situ analysis of Lefty1 (L1) and Lefty2 (L2) (using a common lefty probe; A,B) and Pitx2 (C-H) in Nodal^600/600 embryos (B,D,F,H) and wild-type controls (A,C,E,G). Lateral views with anterior towards left, except for A (posterior view). (A) Lefty1 expression on the left side of the prospective floorplate of the neural tube and Lefty2 in the left LPM in wild-type embryos. (B) Lefty1 is not expressed in Nodal^600/600 mutants, except in a few midline cells close to the node. Lefty2 is expressed in 30% of mutant embryos but at significantly lower levels (left-hand embryo in B). (C) Pitx2 expression in the head and left lateral plate mesoderm (llpm) from the three-somite stage in WT embryos. (D) Nodal^600/600 mutant embryos maintain the anterior expression domain seen in WT embryos, but have a reduced level of expression in the llpm. Note the llpm expression is more posteriorly restricted compared with that in WT. (E) At 8.75 dpc, asymmetric Pitx2 expression in the llpm extends behind the sinoatrial region of the heart (anterior extent of expression indicated by arrowheads). Remnants of the visceral yolk sac (VYS) expressing Pitx2 are seen lateral to the llpm. (F) The anterior extent of Pitx2 expression in the llpm is posteriorly restricted in Nodal^600/600 mutant embryos and is absent from tissue lying behind the sinoatrial region of the heart (arrowheads). (G) After embryonic turning, the difference in the anterior extent of llpm expression of Pitx2 is particularly obvious (compare positions of arrowheads in G and H). The expression in the Nodal^600/600 mutant embryos (H) is anteriorly restricted by approx. two somite widths compared with the wild type (G). This results in Pitx2 not being expressed behind the sinoatrial region of the heart in the mutants. Aberrant heart looping is also obvious within this mutant embryo, although the direction of embryonic turning is normal. Ant, anterior.