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Nodal paralogues underlie distinct mechanisms for visceral left–right asymmetry in reptiles and mammals

Nature Ecology & Evolution volume 4, pages 261–269 (2020)Cite this article

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Abstract

Unidirectional fluid flow generated by motile cilia at the left–right organizer (LRO) breaks left–right (L–R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L–R symmetry breaking. The diversity of mechanisms for L–R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L–R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L–R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.

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Fig. 1: Phylogeny of Nodal1/2 and Cer1/2 genes across vertebrates.
Fig. 2: L–R asymmetric expression of Nodal2 in gecko and turtle embryos.
Fig. 3: Genes required for ciliary motility are not expressed at the blastopore of gecko or turtle embryos.
Fig. 4: Expression in gecko and turtle embryos of genes implicated in L–R patterning in other species.
Fig. 5: Expression of Nodal genes of mammals and chicken in mouse embryos.
Fig. 6: Cilia-dependent and cilia-independent L–R symmetry breaking mechanisms in vertebrates.

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Data availability

All data are available in the main text, the Extended Data and the Supplementary Information. The molecular sequences identified in this study are included in Supplementary Dataset 1.

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Acknowledgements

We thank S. Aizawa for sharing reptile cDNA clones and for advice on reptile embryos, A. Kawasumi for advice on the immunostaining of pSmad2/3 and K. Yamaguchi for technical discussion. This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (17H01435) and by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST) (JPMJCR13W5). U.H. was supported by the Global Science Campus of JST.

Author information

Author notes

  1. Yuichiro Hara

    Present address: Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

Authors and Affiliations

  1. Laboratory for Organismal Patterning, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Eriko Kajikawa, Uzuki Horo, Takahiro Ide, Katsutoshi Mizuno, Katsura Minegishi, Yayoi Ikawa, Hiromi Nishimura & Hiroshi Hamada

  2. SEEDS Program/JST Global Science Campus, Osaka University, Toyonaka, Japan

    Uzuki Horo

  3. NADA Senior High School, Kobe, Japan

    Uzuki Horo

  4. Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Yuichiro Hara & Shigehiro Kuraku

  5. Graduate School for Frontier Biosciences, Osaka University, Suita, Japan

    Masanori Uchikawa

  6. Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Hiroshi Kiyonari

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Contributions

H.H., E.K., U.H. and S.K. designed the project. E.K. performed most experiments with reptile embryos. U.H. performed the initial expression studies. T.I., K.Mizumo and K.Minegishi performed the SEM, TEM and ciliary motility analysis. Y.I. and H.N. generated transgenic mice harbouring various BAC clones. Y.H. and S.K. performed the genomic and phylogenetic analyses. H.K. maintained and provided the gecko animals. M.U. examined Nodal2 expression in chick embryos. H.H. and S.K. wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Shigehiro Kuraku or Hiroshi Hamada.

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Extended data

Extended Data Fig. 1 Detection of structures with features typical of immotile cilia in gecko embryos.

a–c, SEM of gecko embryos at stage 9. The anteroposterior (A-P) and dorsoventral (D-V) axes are indicated. The blastopore (BP) region indicated by the rectangle in a is shown at higher magnification in b. The boxed region in b is shown at higher magnification in c. Cilium-like and microvillus-like structures are indicated by red and white arrowheads, respectively. Scale bars: 100 µm (a), 50 µm (b), or 5 µm (c). d–h, TEM of gecko embryos at stage 9. The boxed regions near the blastopore (BP) in d are shown at higher magnification in e and g. The boxed regions in e and g are shown at higher magnification in f and h, respectively. Both f′ and f′′ are adjacent distal sections of f. The box in f′′ shows one of the doublet microtubules (black arrowhead) at higher magnification. Note that primary cilia with a typical 9+0 microtubule configuration were detected, but that outer dynein arms characteristic of motile cilia were not. Scale bars: 50 µm (d) or 1 µm (e, g).

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Dataset 1.

Reporting Summary

Supplementary Video 1

Movie of the region of the gecko embryo shown in Supplementary Fig. 7b. Note the absence of motile cilia. The images were captured at 150 frames per second for 4 s. Time is shown as minutes:seconds.centiseconds.

Supplementary Video 2

Movie of the region of the gecko embryo shown in Supplementary Fig. 7c. Note that a cilium (arrowhead) is present in this region but does not move. The images were captured at 30 frames per second for 10 s. Time is shown as minutes:seconds.centiseconds.

Supplementary Video 3

Movie of the region of the gecko embryo shown in Supplementary Fig. 7d. Note that a cilium (arrowhead) is present in this region but does not move. The images were captured at 30 frames per second for 10 s. Time is shown as minutes:seconds.centiseconds.

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Kajikawa, E., Horo, U., Ide, T. et al. Nodal paralogues underlie distinct mechanisms for visceral left–right asymmetry in reptiles and mammals. Nat Ecol Evol 4, 261–269 (2020). https://doi.org/10.1038/s41559-019-1072-2

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