Spatiotemporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization

Abstract

SUMMARYAll-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.

Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development

“…Starting the treatment during gastrulation had no detectable effect (not shown). Moreover, H 2 O 2 reduction between 10 and 24 hpf had no macroscopic effect and did not modify GFP expression in specific transgenic lines (mp311b:GFP (Xu et al, 2012); 2.4shh:GFP: ABC#15 (Ertzer et al, 2007); Islet:GFP (Higashijima et al, 2000)) ( Fig. S2A-F).…”

“…Other transgenic lines were used in this study: mp311b:GFP (Xu et al, 2012); 2.4shh:GFP:ABC#15 (Ertzer et al, 2007); Islet:GFP (Higashijima et al, 2000); vmat2:GFP (Wen et al, 2008); p2rx:GFP (Kucenas et al, 2006). The HeLa-HyPer stable cell line in which HyPer expression is controlled by doxycycline was prepared using the HeLa Flp-In cell line that was kindly provided by Stephen Taylor (Tighe et al, 2008).…”

Excitation of Biomolecules with Incoherent Light: Quantum Yield for the Photoisomerization of Model Retinal

“…The importance of retinal photoisomerization has made its mechanism the subject of numerous experimental and computational studies. A related biological process, cis-trans isomerization in Retinoic Acid, has become of increasing interest in its role in zebrafish hindbrain development 9 .…”

Optical control and study of biological processes at the single-cell level in a live organism

“…[170]. Photo-isomerization has also been used to control the activity of retinoic acid[171], whose trans-isoform is a morphogen crucial in the initial stages of development of vertebrates.…”