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New Evidence in Retinoic Acid Debate

Jesse Jenkins

A new imaging technique provides strong evidence regarding retinoic acid’s role in embryonic development. But is this evidence conclusive? Find out...

Retinoic acid (RA) is a signaling molecule synthesized during embryonic development whose role in overseeing the pattern of tissue development has been debated by scientists. Some believe that it helps direct cell fate by forming a two-tailed linear RA gradient along embryo vertebrae, despite a lack of evidence to support that theory.

Researchers have developed a new method to visualize the distribution of RA in a live zebrafish embryo in real-time. Source: RIKEN

But now, researchers at the RIKEN Brain Science Institute in Tokyo, Japan have developed a new method to visualize the distribution of RA in a live zebrafish embryo in real-time. In a paper published online by Nature on April 8, the team describes their observation of two RA concentration gradients moving in opposing directions along the head-to-tail axis of the embryo.

“Shimozono et al. are the first to report a method for in vivo visualization of an endogenous retinoic acid (RA) gradient based on free cytoplasmic RA concentrations,” explained Dennis E. Coyle, an associate professor of anesthesiology at the University of Cincinnati who was not involved in the study. ”This manuscript provides strong yet not conclusive evidence for the presence of a two-tailed linear RA gradient, which has been a subject of continuous debate for many years.”

The study was led by Atsushi Miyawaki, who said that the uncertainty surrounding RA’s role in tissue development was because measuring the concentration of RA is difficult compared to measuring that of other morphogens (molecules that control tissue development).

“All other morphogens are pretty peptidic. So they can observed by fusing them to green fluorescent protein (GFP),” said Miyawaki. “But retinoic acid is not a peptide and it is very hard to label it, so almost all pieces of evidence so far have been indirect.”

To quantify the concentration of retinoic acid in zebrafish embryos during early development, Miyawaki’s lab used a family of genetically encoded probes for RA (GEPRAs) to tag retinoic acid receptors (RARs). To visualize the presence of retinoic acid, the researchers used these GEPRAs and fluorescence resonance energy transfer (FRET), which can determine the distance of two molecules by the interactions of the fluorophores attached to those molecules. In this study, the team used cyan and yellow fluorescent proteins as FRET donors and acceptors.

As a result, the live imaging of zebrafish embryo development revealed a linear concentration gradient of RA in a two-tailed source–sink arrangement across the embryo. “We were really surprised to see a linear gradient,” said Miyawaki.

But Coyle believes that the results don’t quite settle the debate. “In the conclusion, the authors overstate the nature of the visualization by making the assumption that the extracellular concentration, which was never measured, is directly related to the free intracellular concentration of RA,” says Coyle.

For now, Miyawaki’s lab is conducting further tests with GEPRAs and FRET-based techniques and hopes to expand his research to mice as well. “We are now analyzing the mechanism of the diffusion of the retinoic acid, [but] there are many things to do,” said Miyakawai.


1. Shimozono, S., T. Iimura, T. Kitaguchi, S.-i. Higashijima, and A. Miyawaki. 2013. Visualization of an endogenous retinoic acid gradient across embryonic development. Nature advance online publication (April).

Keywords:  microscopy