Chiral blastomere arrangement dictates zygotic left-right asymmetry pathway in snails

Most animals display internal and/or external left-right asymmetry. Several mechanisms for left-right asymmetry determination have been proposed for vertebrates(1-10) and invertebrates(1,2,4,9,11-14) but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaea stagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally(15-18). Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two-or four-cell stage) determines the left-right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four-to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to 'dextralized' sinistral and 'sinistralized' dextral snails-that is, normal healthy fertile organisms with all the usual left-right asymmetries reversed to that encoded by the mothers' genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F-7 congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene( s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left-right patterning of deuterostomes/vertebrates.