FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination

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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Figure 1: Expression of FGF receptors on nodal cilia.
Figure 2: FGF regulates left-specific Ca2+ elevation.
Figure 3: Leftward fluid flow is not affected by SU5402 treatment.
Figure 4: Nodal flow of NVPs is dependent on FGF.
Figure 5: SHH and RA are associated with NVPs.

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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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Correspondence to Nobutaka Hirokawa.

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The authors declare that they have no competing financial interests.

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)

Supplementary Movie Legends (DOC 27 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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