RRC ID 49321
Author Shimozono S, Iimura T, Kitaguchi T, Higashijima S, Miyawaki A.
Title Visualization of an endogenous retinoic acid gradient across embryonic development.
Journal Nature
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 hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. 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.
Volume 496(7445)
Pages 363-6
Published 2013-4-18
DOI 10.1038/nature12037
PII nature12037
PMID 23563268
MeSH Animals Animals, Genetically Modified Body Patterning / physiology Embryo, Nonmammalian / embryology Embryo, Nonmammalian / metabolism Embryonic Development / physiology* Fibroblast Growth Factors / genetics Fibroblast Growth Factors / metabolism Fluorescence Resonance Energy Transfer Gastrula / embryology Gastrula / metabolism HeLa Cells Humans Models, Biological Molecular Probes / analysis Molecular Probes / genetics Molecular Probes / metabolism Molecular Sequence Data Rhombencephalon / embryology Rhombencephalon / metabolism Somites / embryology Somites / metabolism Substrate Specificity Tretinoin / analysis Tretinoin / metabolism* Zebrafish / embryology* Zebrafish / metabolism* Zebrafish Proteins / genetics Zebrafish Proteins / metabolism
IF 42.779
Times Cited 112
DNA material GEPRA-B (RDB15249) GEPRA-G (RDB15250) GEPRA-AA (RDB15251) GEPRA-B/ pT2KXIG deltain (RDB15252) GEPRA-G/ pT2KXIG deltain (RDB15253) GEPRA-AA/ pT2KXIG deltain (RDB15254).