Abstract
The precise specification of left–right asymmetry is an essential process for patterning internal organs in vertebrates. In mouse embryonic development, the symmetry-breaking process in left–right determination is initiated by a leftward extraembryonic fluid flow on the surface of the ventral node. However, it is not known whether the signal transduction mechanism of this flow is chemical or mechanical. Here we show that fibroblast growth factor (FGF) signalling triggers secretion of membrane-sheathed objects 0.3–5 µm in diameter termed ‘nodal vesicular parcels’ (NVPs) that carry Sonic hedgehog and retinoic acid. These NVPs are transported leftward by the fluid flow and eventually fragment close to the left wall of the ventral node. The silencing effects of the FGF-receptor inhibitor SU5402 on NVP secretion and on a downstream rise in Ca2+ were sufficiently reversed by exogenous Sonic hedgehog peptide or retinoic acid, suggesting that FGF-triggered surface accumulation of cargo morphogens may be essential for launching NVPs. Thus, we propose that NVP flow is a new mode of extracellular transport that forms a left–right gradient of morphogens.
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Acknowledgements
We thank P. Tam and colleagues at CMRI for advice on performing embryo dissection and whole-embryo culture. We also thank S. Takeda, R. Takemura, J. Teng, Y. Noda, H. Sato, N. Onouchi, H. Fukuda, M. Sugaya, T. Akamatsu, T. Aizawa and others from the Hirokawa laboratory for providing materials, discussions and technical assistance.This study has been supported by a Center of Excellence Grant-in-Aid (to N.H.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.Author contributions Y.O. helped to produce Fig. 3.
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Supplementary information
Supplementary Movie S1
Leftward nodal flow of fluorescent beads irrespective of FGF signalling. (MOV 1285 kb)
Supplementary Movie S2
Fluorescent images of mouse nodes in ventral views with unidirectional flow of NVPs towards the left. (MOV 822 kb)
Supplementary Movie S3
Ventral view of a mouse node, whose NVP flow is suppressed by 20 µM SU5402. (MOV 940 kb)
Supplementary Movie S4
Ball-throwing release of an NVP from the tip of a dynamically protruding microvillum of the right wall of a node. (MOV 641 kb)
Supplementary Movie S5
Crawling and smashed appearance of NVPs. (MOV 481 kb)
Supplementary Movie S6
Fragmentation of an NVP in the proximity of the left wall. (MOV 111 kb)
Supplementary Movie S7
Restored NVP flow by the addition of SHH-N peptide in the presence of SU5402. (MOV 2932 kb)
Supplementary Movie S8
NVP flow is restored by the addition of 10-7M RA in the presence of SU5402. (MOV 1643 kb)
Supplementary Movie S9
Ventral view of a node treated with SU5402 and IHH-N peptide. (MOV 1057 kb)
Supplementary Movie S10
Ventral view of an iv/iv mutant node. (MOV 2700 kb)
Supplementary Movie S11
Ventral view of a kif3a-/- mutant node lacking the nodal cilia. (MOV 2932 kb)
Supplementary Movie 12
Rotating view of a mouse ventral node fluorescently labelled by 5E1 antibody against SHH-N. (MOV 551 kb)
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Tanaka, Y., Okada, Y. & Hirokawa, N. FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination. Nature 435, 172–177 (2005). https://doi.org/10.1038/nature03494
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DOI: https://doi.org/10.1038/nature03494
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