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Development
of a Method to Control Gene
Expression by Illumination of Ultraviolet Light
Laboratory for Developmental
Gene Regulation |
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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
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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. |
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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. |
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