Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos

Abstract

We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.

Turning heads: Development of vertebrate branchiomotor neurons

“…Knockdown of gene function, using electroporated antisense morpholinos also appears feasible in chick (Kos et al, 2003). In zebrafish, genetic and chemical methods for manipulating gene expression in space and time are available (Scheer and Campos-Ortega, 1999;Halloran et al, 2000;Nasevicius and Ekker, 2000;Ando et al, 2001). Lastly, methods for measuring the electrical activity of BMNs during development are available (Fortin et al, 1995;Abadie et al, 2000;Gust et al, 2003).…”

Development of the zebrafish inner ear

“…The use of caged nucleic acid constructs, which can be photoactivated to give temporal and spatial control of gene expression, may be useful here (Ando et al, 2001). A rapid and potentially powerful way of achieving conditional disruption of protein function is by the application of small molecules; because of its external development and permeability, the zebrafish embryo is well suited to this approach.…”

Constructing the hindbrain: Insights from the zebrafish

“…Forward genetic screens can be targeted to allow isolation of mutants that disrupt very specific processes. New reagents also continue to become available, such as morpholinos to knock-down gene function, and caged RNAs or inducible promoters to allow greater control of ectopic gene expression experiments (Nasevicius and Ekker, 2000;Halloran et al, 2000;Ando et al, 2001). One of the most useful techniques applicable to the zebrafish system is the ability to generate genetic mosaics, and this approach will also continue to provide new information, for example regarding precisely how and when Hox proteins together with their cofactors act to confer regional identity to the hindbrain.…”