The mechanism by which asymmetric signals induce left-right-specific morphogenesis has been elusive. Pitx2 encodes a transcription factor expressed throughout the left lateral plate mesoderm and subsequently on the left side of asymmetric organs such as the heart and gut during organogenesis in the chick embryo. Pitx2 is induced by the asymmetric signals encoded by Nodal and Sonic hedgehog, and its expression is blocked by prior treatment with an antibody against Sonic hedgehog. Misexpression of Pitx2 on the right side of the embryo is sufficient to produce reversed heart looping and heart isomerisms, reversed body rotation, and reversed gut situs.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

The earliest known left-right asymmetric genes are expressed at Hensen's node during chick gastrulation. Gene expression following reorientation of the node shows asymmetry is instructed by adjacent tissue, hence left-right information originates outside the node. Subsequently, the node signals back to the lateral tissue, initiating a cascade leading to left-sided expression of nodal in the lateral plate mesoderm. Loss of nodal expression in the presence of blocking antibodies confirms that Sonic hedgehog is the key signal conveying left-right information from the node; however, manipulation of explant cultures suggests that the induction of nodal requires secondary signals produced in the paraxial mesoderm. These experiments establish the time of action of these signals to and from Hensen's node in establishing left-right asymmetry.

SummaryMany well-known transcriptional regulatory proteins are composed of at least two independently folding domains and, typically, only one of these is a DNAbinding domain. However, some transcriptional regulators have been described that have more than one DNA-binding domain. Regulators with a single DNA-binding domain often bind co-operatively to the DNA in homotypic or heterotypic combinations, and two or more DNA-binding domains of a single regulatory protein can also bind co-operatively to suitably positioned recognition sequences. Here, we examine the behaviour of a chimeric activator of transcription with two different DNA-binding domains, that of the bacteriophage l cI protein and that of the Escherichia coli cyclic AMP receptor protein. We show that these two DNA-binding moieties, when present in the same molecule, can bind co-operatively to a pair of cognate recognition sites located upstream of a test promoter, thereby permitting the chimera to function as a particularly strong activator of transcription from this promoter. Our results show how such a bivalent DNA-binding protein can be used to regulate transcription differentially from promoters that bear either one or both recognition sites.