In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior–posterior axis of structures, including the hindbrain1,2,3,4,5,6and paraxial mesoderm7,8. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes9. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial10. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source–sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.
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The authors thank Y. Wada, R. Aoki, M. Sugiyama, F. Picazo and members of the Brain Science Institute Research Resource Center for technical assistance; C. Yokoyama and A. Terashima for critical reading of the manuscript; the FANTOM Consortium for the cDNA clones; and the Zebrafish International Resource Center for the transgenic zebrafish. This work was partly supported by grants from Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Priority Areas ‘Fluorescence Live Imaging’ and ‘Cell Innovation’ and the Human Frontier Science Program.
The authors declare no competing financial interests.
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Shimozono, S., Iimura, T., Kitaguchi, T. et al. Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496, 363–366 (2013). https://doi.org/10.1038/nature12037
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