INTRODUCTION In vertebrates, the organs of the chest and abdomen have a specific non-random asymmetric arrangement with respect to the midline of the body (situs solitus). The apex of the heart points to the left side, the right and left lung display differences in lobation, the liver is on the right side, the stomach and spleen are on the left, and the large intestine curls from right to left (Moore and Persaud, 1993). Experimental analysis of vertebrate laterality dates back to the 19th century when reversals in asymmetric organ placement (situs inversus) were reported following unilateral warming of chick embryos on the left side (Dareste, 1877). Spemann and his co-workers investigated the origin of body sidedness in amphibians in the early 20th century (reviewed by Wilhelmi, 1921). Three sets of experiments, generation of twinned embryos by ligature, inversion of the middle part of the medullar plate, and unilateral ablations, resulted in defined and predictable laterality defects (Wilhelmi, 1921, and references therein). From these data Wilhelmi concluded that ‘... the left side of the germ has something that the right side does not have’ (Wilhelmi, 1921). This prediction has been confirmed by the discovery of asymmetrically expressed genes at early stages of embryogenesis, prior to morphological asymmetry, both in the lateral plate mesoderm and at the dorsal midline. Gain- and loss-of-function studies in chick and Xenopus have proved the potential of most of these factors to influence laterality. The earliest asymmetric gene activities are found in the chick around the node at the anterior of the primitive streak (activin βB, Levin et al., 1997; cAct-RIIa, HNF3β, shh, nodal, Levin et al., 1995). Later, spatially restricted asymmetric gene expression is found in the right (cSnR; Isaac et al., 1997) and left (nodal; Collignon et al., 1996; Lowe et al., 1996; lefty; Meno et al., 1996) lateral plate mesoderm (LPM) in chick, mouse and Xenopus. Recent work in Xenopus suggests that once bilateral symmetry is broken by an as yet unidentified activity, a left coordinator transmits an instructive signal to the midline. In the frog, processed Vg1 protein can mimic the function of this coordinator (Hyatt and Yost, 1998). In the chick, it is not known what leads to right-sided expression of activin βB, but this in turn establishes asymmetric shh expression in the node. Recent work suggests that left-sided shh acts through an additional unidentified downstream signal to induce the TGFβ signaling molecule nodal in the left LPM (Pagan-Westphal and Tabin, 1998). As the embryonic heart and gut undergo asymmetric looping events, transcription factors (eHAND, and dHAND, Srivastava et al., 1995) as well as components of the extracellular matrix (flectin, Tsuda et al., 1996) and the cytoskeleton (actin, desmin and cytokeratins; Itasaki et al., 1989; Schaart et al., 1989; Price et al., 1996) undergo temporally and spatially restricted activity. Experimental manipulations such as partial loss-of- 1225 Development 126, 1225-1234 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 DEV2373 Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut. Key words: Left-right asymmetry, Pitx2, Homeobox gene, Mouse, Zebrafish, Xenopus SUMMARY The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping Marina Campione 1 , Herbert Steinbeisser 2 , Axel Schweickert 1 , Kirsten Deissler 1 , Frauke van Bebber 3 , Linda A. Lowe 4 , Sonja Nowotschin 1 , Christoph Viebahn 5 , Pascal Haffter 3 , Michael R. Kuehn 4 and Martin Blum 1, * 1 Forschungszentrum Karlsruhe, Institute of Genetics, PO Box 3640, D-76021 Karlsruhe, Germany 2 Abt. V, Department of Cell Biology and 3 Abt. III, Department of Genetics, Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, D-72076 Tübingen, Germany 4 Experimental Immunology Branch, National Cancer Institute, NIH, 10 Center Drive – MSC 1360, Bethesda, MD 20892, USA 5 Anatomisches Institut der Universität Bonn, Nussallee 10, D-53115 Bonn, Germany *Author for correspondence (e-mail: martin.blum@igen.fzk.de) Accepted 22 December 1998; published on WWW 15 February 1999