Retinoic acid is one of the most important signaling molecules during development, and that the embryo gets the right levels of this small molecule is critical. Too much or too little, and the basic patterning of the nervous system and many other organs goes terribly wrong. Indeed, you have to think for a bit to find an organ whose development isn’t affected by retinoic acid levels.
It’s been thought for a long time that retinoic acid acts through a morphogen gradient. However, because the molecule is small, extremely labile, and not protein-based, it’s been difficult to actually measure its levels in the early embryo. The best visualization of a gradient has come from transgenic zebrafish lines. Perz-Edwards et al (2001) established a line in which the expression of YFP is controlled by a trio of retinoic acid response elements (RARE; a regulatory motif that is activated by a complex of retinoic acid and its receptors). This line was nicely quantified by White et al (2007), and in the hindbrain, where retinoic acid is important for anterior-posterior patterning, you can see a gradient of fluorescent signal. However, the signal is only visible from about 22-24 hours of development, well after basic patterning is already established. Even stringing together 12 RAREs to boost the level of fluorescence isn’t sufficient to make the signal visible much earlier (Waxman and Yelon, 2011).
A new paper in Nature by Shimozono and colleagues uses a clever approach to more directly measure retinoic acid levels during zebrafish gastrulation, when the basic patterning of the nervous system and somites is set up. They took the ligand-binding domain of a retinoic acid receptor and fused it with both CFP and YFP. When the ligand-binding domain binds retinoic acid, its conformation changes and there is a FRET event. Measuring FRET therefore gives you a read-out of retinoic acid levels. They show clearly that a two-tailed gradient is established during gastrulation, but the anterior and posterior sides of the gradient (ie. in the hindbrain versus the somites) differ in their dynamics, shape, and regulation.
A useful aspect of their system is that while previous transgenic lines measure the signaling capacity of retinoic acid, as they depend on transcriptional activity, this method measures the molecule’s absolute levels. Comparing quantities and activity could be useful for the study of how retinoic acid is processed, sequestered, and regulated. Another advantage of their system is that they created versions of the sensor protein that have different affinities for retinoic acid- allowing the measurement of both higher and lower levels of the molecule, depending on what part of the embryo you’re interested in.
Overall, this new tool will be extremely valuable to the research community, and will allow labs to study this key signaling molecule more precisely and directly.
Perz-Edwards, A., Hardison, N., Linney, E. (2001) Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Developmental Biology, 229(1):89–101.
Shimozono, S, Iimura, T., Kitaguchi, T, Higashijima, S., Miyawaki, A. Visualization of an endogenous retinoic acid gradient across embryonic development. Nature (2013), published online April 7, 2013.
Waxman, J. and Yelon, D. (2011) Zebrafish retinoic acid receptors function as context-dependent transcriptional activators Developmental Biology, 352(1):128–140.
White, R., Nie, Q., Lander, A., Schilling, T. (2007) Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biology, 5(11): e304.