Laboratory for Developmental Gene Regulation
Study of genetic interactions among genes required for forebrain development by using the spatiotemporal induction of gene expression and knockdown
Lab. for Developmental Gene Regulation, Brain Science Institute, RIKEN
In order to understand the genetic machinery for the establishment of vertebrate forebrain formation, I firstly developed the photo-mediated gene activation technology using 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo)-caged mRNA in zebrafish embryos (Ref. 3-6). Due to its suitability for application of various manipulations in the embryo, analyses of gene function in zebrafish by spatiotemporal control of gene expression may be useful for understanding general mechanisms of vertebrate brain development. As second, I also developed the efficient strategy for determination of genetic epistasis among functionally-related genes in forebrain development by combined use of mRNA caging and antisense morpholino oligonucleotides (AMO) (Ref.2). In summary, when overexpression of gene A in the forebrain by head-restricted uncaging of its mRNA with spot illumination of ultraviolet-light rescued the knockdown effect of gene B, we can consider that gene A acts functionally downstream of gene B. This possibility can be confirmed by observing the absence of rescue potential of mRNA uncaging for gene B to the knockdown effect of gene A. By using this strategy, I had found that a transcriptional regulation factor Lhx2 acts as a major downstream mediator of the function of another transcription factor Six3 in zebrafish forebrain growth (Ref.2). Finally, I developed an alternative method for conditional gene expression and conditional gene knockdown by efficient transfection of plasmid, mRNA and AMO in vivo. I further provided the direct evidence that shows Lhx2 plays an essential role for the generation of telencephalic neurons by this method (Ref.1).
＜Feedbacks from life science society on the achievements＞
caging technology is the first method for efficient spatiotemporal induction of
gene expression in vivo, I have
obtained a great feedback about this achievement from all over the world. Our
workshop meeting for mRNA caging held in 2002 brought together nearly 30
scientists from the foreign countries. In response to the invitation from
University College London, I made a keynote speech on mRNA caging and
construction of mRNA-uncaging system by cooperation with Olympus Europe, Inc.
Recent two publications, “Efficient method for the determination of genetic epistasis by combined use of mRNA caging and AMO” (Ref. 2) and “Efficient transfection strategy for the spatiotemporal control of gene expression” (Ref.1, in press) will also be expected to give considerable impacts to the field of developmental biology.
＜Presentation in invited academic conference＞
Furuta, T. and Okamoto, H. Gene manipulation by photo-sensitive chemical
agents. World Conference on Dosing of Antiinfectives.
＜Presentation in academic conference＞
1. Ando H., Furuta T., Tsien R. Y., and Okamoto H.: “Photo-mediated gene activation using caged RNA in zebrafish embryos”, 5th Int. Conf. on Zebrafish Development and Genetics, (University of Wisconsin-Madison), Madison, USA, June (2002).
2. Ando H., Furuta, T., and Okamoto H.:
“Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos”, 14th
Int. Congr. of Developmental Biology, (The Japanese Society of Developmental
3. Okamoto H. and Ando H.: “Epistatic
analysis of gene regulation in zebrafish brain development using caged mRNA
technology”, 23rd Ann. Meet. of Australian Neuroscience Society,
4. Okamoto H. and Ando H.: “Epistatic analysis of gene regulation in zebrafish brain development using caged mRNA technology”, 23rd Ann. Meet. of Australian Neuroscience Society Satellite Meetings: Regulation of Expression in the Brain, Heron Island, Australia, Jan. (2003).
＜List of main achievements in present laboratory＞
1. Ando,H. and Okamoto,H. Efficient transfection strategy for the spatiotemporal
control of gene expression in zebrafish. Marine Biotechnology, (in press)
2. Ando,H., Kobayashi,M., .Tsubokawa,T., Oyemura,K., Furuta,T. and Okamoto,H. Lhx2 mediates the activity of Six3 in zebrafish forebrain growth, Developmental Biology, 287, 456-468(2005)
3. Ando,H. and Okamoto,H. Photo-mediated gene activation by using caged mRNA in zebrafish embryos, Methods in Cell Biology, 77, 159-171(2004)
4. Okamoto,H., Hirate,Y. and Ando,H. Systematic identification of factors in zebrafish regulating the early midbrain and cerebellar development by ordered differential display and caged mRNA technology, Frontiers in Bioscience, 9, 93-99(2004).
5. Ando,H. and Okamoto,H. Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA, Methods in Cell Science, 25, 25-31(2003).
6. Ando,H., Furuta,T., Tsien, R..Y., and Okamoto,H. Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos, Nature Genetics, 28, 317-325(2001).
1. Full length sequence of zebrafish lhx2 cRNA: Genbank accession number; AB188255)
2. Full length sequence of zebrafish lhx9 cDNA: Genbank accession number, AB188254)
1. “Chemical synthesis of 6-bromo-4-diazomethyl-7-hydroxycoumarine (Bhc-diazo)”
Licensed to Wako Pure Chemical Industry Inc. (2005)
2. “Method for gene expression control” Application No. : TOKUGANN 2001-124452