Hideki Ando

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

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.

Photo-mediated gene activation by using caged mRNA in zebrafish embryos.

Publisher Summary This chapter focuses on photo-mediated gene activation by using caged mRNA in zebrafish embryos. To apply the RNA-caging technology for controlling gene expression at spatially and temporally high resolution in rapidly developing zebrafish embryos, it is essential to use a caging agent that is easy to react with mRNA in vitro and to be removed in vivo by a minimum amount of photoillumination. To meet this requirement, an RNA caging system that uses the caging agent 6-bromo-4-diazomethyl-7-hydroxycoumarin is developed. Caged compounds that can be activated by photoirradiation should offer an ideal method to control the intracellular concentration of a signaling molecule and provide an opportunity to control gene expressions with high temporal and spatial resolution. Among the caging groups available, the Bhc group is chosen to mask the translational activity of mRNA. Synthesis of caged RNA can be achieved in two ways. In the first, a full-length mRNA is used as the starting material and the synthesis involves a chemoselective protection of one of the functional groups in the RNA with a caging agent. In the second, a caged nucleoside monomer is the precursor for a chemical synthesis of the caged full-length mRNA. The latter approach requires the rather complicated synthesis of an appropriately protected caged monomer, and, at present, the chemical synthesis can only be applied to short RNAs.

Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA

We previously reported mRNA caging technology as a novel and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos and as an alternative to the gene knockdown approach using antisense morpholino oligonucleotides. The caging reagent used is 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo), which forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA. Mainly because of the reduced solubility of caged mRNA in aqueous solutions, special care in handling is needed. The Bhc-diazo group binds to the phosphate moieties of RNA and abolishes the translational activity of the latter. The translational activity of Bhc-caged mRNA is restored by photolysis/uncaging when exposed to long-wave UV light (350 approximately 365 nm). In this paper we describe the technique and detailed procedures for spatially and temporally controlled induction of gene expression in zebrafish embryos.

Efficient Transfection Strategy for the Spatiotemporal Control of Gene Expression in Zebrafish

Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.

Systematic identification of factors in zebrafish regulating the early midbrain and cerebellar development by odered differential display and caged mRNA technology.

During brain development, various transcription factors are activated in the regional specific manner and define the identities characteristic to individual regions, and many of such factors have been identified in the vertebrate brain, by taking advantage of the structural and functional conservation of them with the invertebrate counterparts. However, it is still largely unknown why individual transcription factors can define the final morphology and function of the tissues expressing these factors because of the lack of knowledge on which genes are actually up- or down-regulated as downstream targets of individual transcription factors. In this review, we introduce novel technologies which we have invented or improved as a part of our endeavor to identify and functionally analyze the downstream target genes of Isle-3 which are involved in development of the midbrain and the midbrain/hindbrain boundary region in zebrafish embryos. Our strategy and technologies can be applied to analyzing the downstream genes of any other transcription factors.