Nodal signalling and the roles of the transcription factors SnR and Pitx2 in vertebrate left-right asymmetry.

Left-specific mesodermal expression of the homeobox gene Pitx2 links a gastrula-stage intercellular signalling cascade to the later development of vertebrate left-right organ asymmetry through the Nodal signalling pathway [1] [2][3] [4][5] [6]. SnR (Drosophila snail-related), a gene conserved in vertebrates and encoding a zinc-finger protein, appears to function in a similar manner to Pitx2, but through right-specific mesodermal expression [7] [8]. Here, we present direct evidence for an earlier proposal [7] that Nodal signalling specifically represses expression of SnR in left lateral mesoderm, and thus ensures its normal confinement to the right, while activating Pitx2 on the left. We addressed the relationship between Pitx2 and SnR using antisense disruption of SnR. Relatively severe antisense-SnR treatment led to massive ectopic expression of Pitx2 on the right, accompanied by randomisation of situs with embryos showing aspects of left-cardiac isomerism. This indicates a gene cascade relationship in the propagation of left-right information, whereby nodal activates Pitx2 on the left through a double-negative mechanism involving the repression of SnR's repressor role on Pitx2. Milder antisense-SnR treatment reversed heart-loop direction and embryo torsion at significant incidence, although Pitx2 expression remained normally left-confined throughout. This reversed morphology following SnR disruption alone appeared different from that in which additional ectopic right-hand Pitx2 expression was seen. Therefore, in addition to their regulatory gene cascade relationship, these two transcription factors appear to have further, parallel but non-redundant, roles in directly controlling normally right- and left-specific morphogenetic processes.