Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Stern, C. D. & Foley, A. C. Molecular dissection of Hox gene induction and maintenance in the hindbrain. Cell 94, 143–145 (1998)
Begemann, G., Marx, M., Mebus, K., Meyer, A. & Bastmeyer, M. Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain. Dev. Biol. 271, 119–129 (2004)
Maves, L. & Kimmel, C. B. Dynamic and sequential patterning of the zebrafish posterior hindbrain by retinoic acid. Dev. Biol. 285, 593–605 (2005)
Sirbu, I. O., Gresh, L., Barra, J. & Duester, G. Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression. Development 132, 2611–2622 (2005)
Hernandez, R. E., Putzke, A. P., Myers, J. P., Margaretha, L. & Moens, C. B. Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development. Development 134, 177–187 (2007)
White, R. J., Nie, Q., Lander, A. D. & Schilling, T. F. Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biol. 5, e304 (2007)
Diez del Corral, R. et al. Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65–79 (2003)
Moreno, T. A. & Kintner, C. Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis. Dev. Cell 6, 205–218 (2004)
Aulehla, A. & Pourquié, O. Signaling gradients during paraxial mesoderm development. Cold Spring Harb. Perspect. Biol. 2, a000869 (2010)
White, R. J. & Schilling, T. F. How degrading: Cyp26s in hindbrain development. Dev. Dyn. 237, 2775–2790 (2008)
Kudoh, T., Wilson, S. W. & Dawid, I. B. Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129, 4335–4346 (2002)
Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971–980 (2000)
Entchev, E. V., Schwabedissen, A. & Gonález-Gaitán, M. Gradient formation of the TGF-β homolog Dpp. Cell 103, 981–991 (2000)
Gregor, T., Wieschaus, E. F., McGregor, A. P., Bialek, W. & Tank, D. W. Stability and nuclear dynamics of the bicoid morphogen gradient. Cell 130, 141–152 (2007)
Yu, S. R. et al. Fgf8 morphogen gradient forms by a source–sink mechanism with freely diffusing molecules. Nature 461, 533–536 (2009)
Müller, P. et al. Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system. Science 336, 721–724 (2012)
Rhinn, M. & Dollé, P. Retinoic acid signalling during development. Development 139, 843–858 (2012)
Crick, F. Diffusion in embryogenesis. Nature 225, 420–423 (1970)
Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47 (1969)
Eichele, G. & Thaller, C. Characterization of concentration gradients of a morphologically active retinoid in the chick limb bud. J. Cell Biol. 105, 1917–1923 (1987)
Perz-Edwards, A., Hardison, N. L. & Linney, E. Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Dev. Biol. 229, 89–101 (2001)
Emoto, Y., Wada, H., Okamoto, H., Kudo, A. & Imai, Y. Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish. Dev. Biol. 278, 415–427 (2005)
Sawada, A. et al. Fgf/MAPK signaling is a crucial positional cue in somite boundary formation. Development 128, 4873–4880 (2001)
Dubrulle, J., McGrew, M. J. & Pourquié, O. FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232 (2001)
Draper, B. W., Morcos, P. A. & Kimmel, C. B. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30, 154–156 (2001)
Brand, M. et al. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123, 179–190 (1996)
Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain–hindbrain boundary development and somitogenesis. Development 125, 2381–2395 (1998)
Hamade, A. et al. Retinoic acid activates myogenesis in vivo through Fgf8 signalling. Dev. Biol. 289, 127–140 (2006)
Napoli, J. L. Interactions of retinoid binding proteins and enzymes in retinoid metabolism. Biochim. Biophys. Acta 1440, 139–162 (1999)
Haugland, R. P. & Johnson, I. D. in Fluorescent and Luminescent Probes for Biological Activity (ed. Mason, W. T. ) 40–50 (Academic, 1999)
Carninci, P. et al. The transcriptional landscape of the mammalian genome. Science 309, 1559–1563 (2005)
Zhang, Z. P. et al. Role of Ser289 in RARγ and its homologous amino acid residue of RARα and RARβ in the binding of retinoic acid. Arch. Biochem. Biophys. 380, 339–346 (2000)
Sawano, A. & Miyawaki, A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 15, e78 (2000)
Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genesis 174, 639–649 (2006)
Gustafson, A. L., Donovan, M., Annerwall, E., Dencker, L. & Eriksson, U. Nuclear import of cellular retinoic acid-binding protein type I in mouse embryonic cells. Mech. Dev. 58, 27–38 (1996)
Dong, D., Ruuska, S. E., Levinthal, D. J. & Noy, N. Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J. Biol. Chem. 274, 23695–23698 (1999)
Norris, A. W., Cheng, L., Giguère, V., Rosenberger, M. & Li, E. Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration. Biochim. Biophys. Acta 1209, 10–18 (1994)
Acknowledgements
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.
Author information
Authors and Affiliations
Contributions
S.S. and A.M. conceived and designed the study. S.S. performed all the experiments, analysed the data and designed the manuscript. T.I. supervised the experiments on somitogenesis. T.K. and S.H. generated transgenic zebrafish lines. A.M. designed and wrote the manuscript, and supervised the project.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
This file contains Supplementary Figures 1-13 and Supplementary References. (PDF 2177 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature12037
This article is cited by
-
Highlighting the gaps in hazard and risk assessment of unregulated Endocrine Active Substances in surface waters: retinoids as a European case study
Environmental Sciences Europe (2021)
-
Generation of extracellular morphogen gradients: the case for diffusion
Nature Reviews Genetics (2021)
-
Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics
Scientific Reports (2021)
-
Optogenetic stimulation inhibits the self-renewal of mouse embryonic stem cells
Cell & Bioscience (2019)
-
Hindbrain induction and patterning during early vertebrate development
Cellular and Molecular Life Sciences (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.