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Visualization of an endogenous retinoic acid gradient across embryonic development

机译：胚胎发育中内源视黄酸梯度的可视化

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摘要

“形态发生素”是可扩散的信号作用分子，根rn据细胞的浓度为其安排不同的命运。“视黄rn酸”是一种非典型的“形态发生素”——一种亲rn脂性分子，而不是一种多肽。大多数“形态发rn生素”可以通过将它们融合到荧光标记中来存rn活体中观测，但非肽“视黄酸”却难以直接成rn像，而且目前仍不清楚它在胚胎中是怎样在空rn间上分布的。现在，Atsushi Miyawaki及其同rn事研制出了基因编码的探针，被称为GEPRAs，rn它们允许在活体中对“视黄酸”浓度进行定量rn测定。他们发现，沿斑马鱼胚胎的前后轴线rn存在“视黄酸”的线性浓度梯度 同时他们也rn得出结论认为，这些数据支持Francis Crick存rn1970年提出的有关“形态发生素”动态的源—rn汇模型。%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.
机译：“形态发生素”是可扩散的信号作用分子，根rn据细胞的浓度为其安排不同的命运。“视黄rn酸”是一种非典型的“形态发生素”——一种亲rn脂性分子，而不是一种多肽。大多数“形态发rn生素”可以通过将它们融合到荧光标记中来存rn活体中观测，但非肽“视黄酸”却难以直接成rn像，而且目前仍不清楚它在胚胎中是怎样在空rn间上分布的。现在，Atsushi Miyawaki及其同rn事研制出了基因编码的探针，被称为GEPRAs，rn它们允许在活体中对“视黄酸”浓度进行定量rn测定。他们发现，沿斑马鱼胚胎的前后轴线rn存在“视黄酸”的线性浓度梯度 同时他们也rn得出结论认为，这些数据支持Francis Crick存rn1970年提出的有关“形态发生素”动态的源—rn汇模型。%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.

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  • 来源
    《Nature》 |2013年第7445期|363-366259261|共6页
  • 作者

    Satoshi Shimozono; Tadahiro Iimura; Tetsuya Kitaguchi; Shin ichi Higashijima; Atsushi Miyawaki;

  • 作者单位

    Laboratory for Cell Function Dynamics. Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan;

    Laboratory for Cell Function Dynamics. Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan;

    Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan;

    Nationa Institutes of Natural Sciences, Okazaki Institute for integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan;

    Laboratory for Cell Function Dynamics. Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan,Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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