Development of a Method to Control Gene Expression by Illumination of Ultraviolet Light Laboratory for Developmental Gene Regulation Since zebrafish mbryos are transparent and have a comparatively simple nervous system, they are extremely suitable for use in studies of the basic mechanisms of vertebrate brain development (Figs.1 A, B). In the spinal cord of zebrafish embryos at 20 hours after fertilization, there are only six types of nerve cells per hemisegment (one side of the spinal segment);three or four motor neuronal cells (neurons), two to four intermediating neurons and one to three sensory neurons), total of approximately 11 neurons at most. These neurons extend their axons in predetermined directions (Fig.1C). For example, for the three motor neurons present in each hemisegment, RoP (rostral primary), MiP (middle primary) and CaP (caudal primary), each name corresponding to a position of the cell body in the hemisegment. CaP is always connected with the ventral side of the segmental body trunk muscle, MiP with the dorsal side, and RoP with the septal side. We are currently studying the mechanisms underlying the behavioral differences of each neuron. To date, we have found for example, that in a zebrafish embryo, transcription factors Islet-1 and a similar one, Islet-2, are specifically expressed only in RoP and CaP, respectively (Fig. 1D). After discovering such a stunning expression pattern as an alternating expression in neighboring neurons, we were attracted to knowing how the characteristic of neurons would be affected if we disrupted this expression pattern. Taking advantage of transparency of the zebrafish embryos, we considered that it would be possible to induce gene expression by illuminating with small light spot, which is approximately one cell size. The chemical compound 6-Bromo-4-diazomethyl-7-hydroxycoumarine(Bhc-diazo), developed by Furuta and Tsein, was a key ingredient as we proceeded with the investigation. We found that this compound binds to the phosphate group of DNA and RNA, and further that this compound can be released again by irradiation with 365-nm wavelength ultraviolet light over a short period of time (Fig. 2A). By injecting mRNA for Green Fluorescent Protein (GFP), which is extracted from jelly fish and caged with Bhc-diazo in advance, into the zebrafish embryo at its one-cell stage, and then applying spot ultraviolet light only to the head of the embryo at 12 hours after fertilization (Fig. 2B), GFP was translated only in the head, and only the head became fluorescent (Fig. 2C). We called this technique the "caged mRNA technique". It is known that the binding of Bhc with phosphate groups can be released not only by absorption of a single photon at a 365 nm wavelengh, but also by a simultaneous collision with two photons at a 730 nm wavelengh (infrared ray), which have twice the wavelengh and half the energy of the former. A special laser, called a mode-lock laser, can generate such a high-density impulses of photons. Even with this laser, simultaneous collision of two photons with the target molecule (bond Bhc) can take place only around the focal point of the light from the objective lens, enabling gene activation by removal of Bhc only in the cells in the closest vicinity of the focal point. With this technological improvement, we are now aiming at proceeding in our research toward the control of gene expression at a one-cell level. nature genetics 28; 317-325, 2001 Hideki Ando1,3,Toshiaki Furuta2,4, and Hitoshi Okamoto1,3, 1: Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute 2: Department of Biomolecular Science, Faculty of Science, Toho University (furuta@biomol.sci.toho-u.ac.jp) 3: CREST, Japan Science and Technology Corporation 4: PREST, Japan Science and Technology Corporation 　 Fig. 1 Extension of axons from neurons in the spinal cord of zebrafish embryos. A: adult zebrafish. B: Zebrafish embryos at 20 hours after fertilization. C: Schematic illustration of neurons in the spinal cord of zebrafish embryos at 24 hours after fertilization. D: Expression patterns of Islet-1 mRNA (red) and Islet-2 mRNA (violet) in the spinal cord of zebrafish embryo. Islet-1 mRNA is expressed in RoP and Islet-2 mRNA is expressed in CaP. Both mRNAs are expressed in the Rohon-Beard neurons. 　 Fig. 2 Regulation of gene activation using the mRNA caging method. A: Binding of Bhc-diazo with mRNA and mechanism of its release using irradiation with ultraviolet light. B: Schematic illustration of irradiation with ultraviolet light on the head of zebrafish embryos at 12 hours after fertilization. C: Induction of head-specific expression of GFP in the embryo, in which caged GFP mRNA was injected at the one-cell stage and then only the head was irradiated with ultraviolet light at 12 hours after fertilization.