Nature © Macmillan Publishers Ltd 1998 8 Pitx2 determines left–right asymmetry of internal organs in vertebrates Aimee K. Ryan*†, Bruce Blumberg†‡, Concepcio´ n Rodriguez-Esteban†‡, Sayuri Yonei-Tamura†‡, Koji Tamura‡, Tohru Tsukui‡, Jenniferde la Pen˜ a‡, Walid Sabbagh‡, Jason Greenwald‡, Senyon Choe‡, Dominic P. Norris§, Elizabeth J. Robertson§, Ronald M. Evans‡k, Michael G. Rosenfeld* & Juan Carlos Izpisu´ a Belmonte‡ * Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0648, USA § Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA k Howard Hughes Medical Institute, ‡ The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037, USA † These authors contributed equally to this work ........................................................................................................................................................................................................................................................ The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left–right asymmetry in vertebrates. The vertebrate body exhibits bilateral symmetry externally whereas the internal organs display significant left–right asymmetry. During organogenesis, the unpaired organs of the chest and abdomen begin development in the midline and then lateralize, with the first morpho- logical markers of left–right asymmetry being the right-sided looping of the developing heart. A second sign of asymmetry is then mani- fested by the rotation of the body in amniote embryos. Virtually all visceral organs ultimately show left–right asymmetry, either with respect to their location in the body cavity or by morphological differences on one side versus the other. The left–right asymmetries of internal organ placement are invariant within a given species and have been conserved throughout evolution. Normal organ placement is termed situs solitus, and the mirror-image arrangement is situs inversus. Other defects of situs are partial (heterotaxy) or complete (isomerism) loss of asymmetry. Left–right axis malformations in humans are phenotypically variable and genetically heterogeneous 1,2 . Generally, individuals with complete situs inversus do not suffer severe clinical consequences, whereas heterotaxia and isomerism are associated with moderate-to-severe physiological complications 3,4 . As the establishment of correct left–right asymmetry is critical for survival, the mechanisms governing initiation and maintenance of these asymmetries should be tightly regulated and evolutionarily conserved. Several models have been proposed to account for these asymmetries (reviewed in refs 5–7). In chick, there is a signalling cascade involving members of the TGF- superfamily, namely activin-B and Nodal, the activin receptor RIIA (cAct-RIIA) and Sonic hedgehog (Shh), all of which are asymmetrically expressed with respect to the left–right axis 8,9 . Activin-bB, present asymme- trically on the right side of stage 3–5+ embryos 9,10 , is thought to induce local expression of cAct-RIIA 8,10 , which in turn represses the bilaterally symmetrical Shh expression in Hensen’s node on the right 8,9 . This leads to left-sided expression of Shh and induction of nodal in the left lateral plate mesoderm 8 . Misexpression of activin or Shh disrupts the normal expression pattern of nodal and rando- mizes heart looping. In Xenopus, inappropriate expression of the TGF- family member Vg-1 inverts nodal expression and results in situs inversus 11,12 . In contrast to the chicken model, targeted gene deletion of Shh, activin-B, follistatin or Act-RIIA in mice does not alter the left–right orientation of the heart or of the internal organs, calling into question their role in left–right patterning in the mouse 13–17 . Mice null for Act-RIIB, which is not asymmetrically expressed in chick or mouse, exhibit defects in left–right asymme- tries, including isomerisms 18 , suggesting that Act-RIIB is a critical component of the left–right pathway in mouse. Of the many molecules that have been implicated in left–right signalling during vertebrate embryogenesis, only Nodal exhibits a clear correlation between its expression in the lateral plate meso- derm and visceral situs 19,20 . In inv/inv mice, where virtually all animals exhibit situs inversus, nodal is expressed only in the right lateral plate mesoderm 19,20 . In iv mice, where left–right development is randomized, all four possible patterns of nodal expression are observed: left, right, bilateral and absent 20 (see also ref. 21). nodal expression is bilateral in Fused toes 22 and no turning 23 mice, which also have randomized left–right asymmetries. Altering the normal nodal expression pattern in the left lateral plate mesoderm in Xenopus and chick is also associated with changes in left–right development 8,11,24–26 . Thus, Nodal appears to be a conserved factor in the cascade that establishes left–right asymmetry in all verte- brates. The observations that nodal expression reliably predicts situs and that loss of Act-RIIB function leads to defects in situs suggests that these factors function in a common signalling pathway. Although progress has been made in understanding early events in the determination of left–right asymmetry, much is yet to be learned about how multiple extracellular signals are transduced, propagated and maintained, ultimately leading to visceral asym- metry. Transcription factors are good candidates for mediating these processes. However, relatively little is known of their role in this process, and only three have been implicated in the left–right asymmetry pathway. HNF-3 may have a role because it is transiently asymmetrically expressed in the chick 8 and because HNF-3b +/- , nodal laZ/+ double-heterozygous mice express lacZ bilat- erally in the lateral plate mesoderm and have defects in the positioning of the viscera and heart, and random embryonic rotation 19 . The zinc-finger gene Snail-Related (cSnR) which is initially expressed articles NATURE | VOL 394 | 6 AUGUST 1998 545