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

Abstract

Most animals display internal and/or external left–right asymmetry. Several mechanisms for left–right asymmetry determination have been proposed for vertebrates1,2,3,4,5,6,7,8,9,10 and invertebrates1,2,4,9,11,12,13,14 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 maternally15,16,17,18. 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 F7 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.

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Figure 1: Reversal of the third cleavage directions by micromanipulation and the resultant 8-, 12- and 16-cell stage embryos.
Figure 2: Chirality-reversed eight-cell stage embryos developed to snails with an oppositely-coiled shell and visceral situs inversus.
Figure 3: nodal and Pitx expression in control, congenic F 7 progeny, and chirality-inverted L. stagnalis embryos.
Figure 4: Determinants of chirality in the snail L. stagnalis.

Accession codes

Data deposits

Sequences of L. stagnalis nodal and Pitx are deposited at GenBank, with accession numbers respectively GU073383 and GU073384.

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Acknowledgements

We thank G. Smit for his gift of dextral and sinistral stock of L. stagnalis. We also thank K. Miyoshi, Y. Ozawa and H. Kuwata of the Kuroda Chiromorphology team for their help in rearing snails and creating F7 congenic snails. K. Fujikura, A. Okubo and G. Sai are thanked for their preliminary attempts at in situ hybridization experiments.

Author Contributions R.K. conceived the study, designed/coordinated the experiments and wrote the manuscript. B.E. performed the reversal experiments and whole mount in situ hybridization (WISH) on reversed embryos. M.A. performed WISH on control and F7 congenic snails. M.S. cloned and characterized nodal and Pitx from L. stagnalis to make template vector for the WISH probes. B.E. and M.S. provided comments on the manuscript.

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Correspondence to Reiko Kuroda.

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Kuroda, R., Endo, B., Abe, M. et al. Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails . Nature 462, 790–794 (2009). https://doi.org/10.1038/nature08597

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