A pathway to asymmetry

Vertebrates appear bilaterally symmetrical. Beneath the surface, however, their internal organs display marked left/right (L/R) asymmetry. To achieve this final body plan, vertebrate embryos must distinguish right from left and transmit this information to individual organs as they develop. How are these early L/R patterning decisions made?

A key breakthrough came in 1995 through the pioneering work of Mike Levin, Cliff Tabin and colleagues, who discovered a cascade of genes regulating L/R organ asymmetry in the chick. The starting point for this work was the discovery of a transient asymmetry in Shh expression on the left side of Hensen's node — a midline structure that serves as a key source of patterning signals in the early embryo. Subsequently, Levin et al. stumbled on a second gene, Nodal, that was expressed immediately adjacent to Shh in a domain extending throughout the embryo's left side. Importantly, when Levin et al. misexpressed Shh on the right side of the embryo, Nodal was induced ectopically and the normal rightward bending of the heart tube became randomized. This study revealed, for the first time, the existence of genes with L/R asymmetric expression patterns that could influence the direction of subsequent L/R morphogenetic events.

In 1996, follow-up studies led by Liz Robertson and Michael Kuehn showed that this asymmetry in Nodal expression is also present in mouse and Xenopus, identifying Nodal as a central player in a conserved vertebrate L/R pathway. Simultaneously, Hiroshi Hamada and colleagues discovered another gene, which they called Lefty because of its striking expression in the left lateral plate mesoderm and in the left half of the prospective floor plate. The Lefty locus was subsequently found to contain two closely linked and highly related genes, Lefty-1 and Lefty-2.

The overlapping expression of Nodal and Lefty-2 in the left lateral plate mesoderm indicated that these genes might act together to impart left-sided identity to developing organs. However, an unexpected role for Lefty genes subsequently emerged from the analysis of Lefty-1 knockout mice: Lefty-1, rather than specifying 'leftness', is actually required for main-taining right-sided identity. As the primary site of Lefty-1 expression is in the left half of the prospective floor plate, Meno et al. proposed that Lefty-1 acts as a midline barrier, preventing left-sided signals, such as Nodal, from crossing the midline and conferring left identity to cells on the right.

Nodal expression is extinguished before the onset of L/R morphogenesis, so how does it influence organ situs? In 1998, studies led by Cliff Tabin, Juan Carlos Izpisúa Belmonte, Sumihare Noji and Marian Ros identified Pitx2 as a key downstream mediator in the L/R pathway. Unlike Nodal, Pitx2 expression is maintained on the left side as organs develop, bridging the gap between asymmetric gene expression and L/R morphogenesis.

Still, a key question remained. How is the initial distinction between left and right made? In a landmark 1998 study, Nobutaka Hirokawa and colleagues made the remarkable discovery that the mouse node contains a cluster of motile cilia which generate a leftward flow across the node's ventral surface. As the node is where the earliest L/R asymmetric expression patterns are seen, and directional flow could, in principle, arise from the inherent chirality of the ciliary apparatus without prior input of L/R information, these results indicated that ciliary motion might initiate L/R axis specification.

So, in the space of 3 exciting years, we witnessed the birth of a model that could explain, in broad terms, how embryos distinguish left from right and use this information to coordinate the asymmetric shape and positioning of organs along the body's L/R axis.