LEFT RIGHT PATTERNING SIGNALS AND THE INDEPENDENT REGULATION OF DIFFERENT ASPECTS OF SITUS IN THE CHICK-EMBRYO/

Recently, a pathway of genes which are part of a cascade regulating th e side on which the heart forms during chick development was character ized (M. Levin ct al., 1995, Cell 82, 1-20). Here we extend these prev ious studies, showing that manipulation of at least one member of the cascade, Sonic hedgehog (Shh), can affect the situs of embryonic rotat ion and of the gut, in addition to the heart. Bilateral expression of Shh, which is normally found exclusively can the left, does not result in left isomerism (a bilaterally symmetrical embryo having two left s ides) nor in a complete situs inversus phenotype. Instead, misexpressi on of Shh on the right side of the node, which in turn leads to bilate ral nodal expression, produces a heterotaxia-like condition, where dif ferent aspects of laterality are determined independently. Heart situs has previously been shown to be altered by ectopic Shh and activin. H owever, the most downstream gene identified in the LR pathway, nodal, had not been functionally linked to heart laterality. We show that ect opic (right-sided) nodal expression is able to affect heart situs, sug gesting that the randomization of heart laterality observed in Shh and activin misexpression experiments is a result of changes in nodal exp ression and that nodal is likely to regulate heart situs endogenously. The first defined asymmetric signal in the left-right patterning path way is Shh, which is initially expressed throughout Hensen's node but becomes restricted to the left side at stage 4(+). It has been hypothe sized that the restriction of Shh expression 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 repress Shh on the left side of the node, as well as to induce ectopic expression of a normally right-sid ed marker, the activin receptor cAct-RIIa. Here we provide further evi dence in favor of this model. We find that a member of this family, Ac tivin PB, is indeed expressed asymmetrically, only on the right side o f Hensen's node, at the correct time for it to be the endogenous asymm etric activin signal. Furthermore, we show that application of follist atin-loaded beads eliminates the asymmetry in Shh expression, consiste nt with an inhibition of an endogenous member of the activin-BMP super family. This combined with the previous data on exogenous activin supp orts the model that Activin beta B functions in the chick embryo to in itiate Shh asymmetry. While these data extend our understanding of the early signals which establish left-right asymmetry, they leave unansw ered the interesting question of how the bilateral symmetry of the emb ryo is initially broken to define a consistent left-right axis. Analys is of spontaneous chick twins suggests that, whatever the molecular me chanism, left-right patterning is unlikely to be due to a blastodermal prepattern but rather is initiated in a streak-autonomous manner. (C) 1997 Academic Press.