Left/Right Patterning Signals and the Independent Regulation of Different Aspects ofSitusin the Chick Embryo

Abstract

Recently, a pathway of genes which are part of a cascade regulating the side on which the heart forms during chick development was characterized (M. Levinet al.,1995,Cell82, 1–20). Here we extend these previous studies, showing that manipulation of at least one member of the cascade,Sonic hedgehog(Shh), can affect thesitusof embryonic rotation and of the gut, in addition to the heart. Bilateral expression ofShh,which is normally found exclusively on the left, does not result in left isomerism (a bilaterally symmetrical embryo having two left sides) nor in a completesitus inversusphenotype. Instead, misexpression ofShhon the right side of the node, which in turn leads to bilateralnodalexpression, produces a heterotaxia-like condition, where different aspects of laterality are determined independently. Heartsitushas previously been shown to be altered by ectopic Shh and activin. However, the most downstream gene identified in the LR pathway,nodal,had not been functionally linked to heart laterality. We show that ectopic (right-sided)nodalexpression is able to affect heartsitus,suggesting that the randomization of heart laterality observed inShhandactivinmisexpression experiments is a result of changes innodalexpression and thatnodalis likely to regulate heartsitusendogenously. The first defined asymmetric signal in the left–right patterning pathway isShh,which is initially expressed throughout Hensen's node but becomes restricted to the left side at stage 4+. It has been hypothesized that the restriction ofShhexpression may be due to repression by an upstream activin-like factor. The involvement of such an activin-like factor on the right side of Hensen's node was suggested because ectopic activin protein is able to repressShhon the left side of the node, as well as to induce ectopic expression of a normally right-sided marker, the activin receptorcAct-RIIa.Here we provide further evidence in favor of this model. We find that a member of this family,Activin βB,is indeed expressed asymmetrically, only on the right side of Hensen's node, at the correct time for it to be the endogenous asymmetric activin signal. Furthermore, we show that application of follistatin-loaded beads eliminates the asymmetry inShhexpression, consistent with an inhibition of an endogenous member of the activin–BMP superfamily. This combined with the previous data on exogenous activin supports the model thatActivin βBfunctions in the chick embryo to initiateShhasymmetry. While these data extend our understanding of the early signals which establish left–right asymmetry, they leave unanswered the interesting question of how the bilateral symmetry of the embryo is initially broken to define a consistent left–right axis. Analysis of spontaneous chick twins suggests that, whatever the molecular mechanism, left–right patterning is unlikely to be due to a blastodermal prepattern but rather is initiated in a streak-autonomous manner.