高橋 将文

Electroporation has been widely used in various animals to introduce transgenes into their tissues and organs. We have previously developed a gene transfer method into the developing brain of rat and mouse embryos by applying electroporation to a mammalian whole embryo culture technique. We can directly transfer exogenous genes and small nucleic acids, e.g., double strand RNAs, siRNAs, and Morpholino oligos, into desirable regions of the developing brain of cultured embryos at different stages by easily adjusting the direction of electrodes. This method enables us to provide simple and convenient gain-of- function and loss-of-function studies to explore novel understanding of molecular and cellular mechanisms underlying mammalian brain development.

Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell--cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use. Whole-embryo mammalian culture system was established by New and colleague in the 1970s, and was modified thereafter by several researchers (reviewed by New, 1971, 1978 Sturm and Tam, 1993 Hogan et al., 1994 Eto and Osumi-Yamashita, 1995 Tam, 1998). At first, the whole-embryo culture system was used in the field of teratology, and then applied in a variety of developmental biology fields, because this system well maintains the embryonic growth and morphogenesis that are comparable to those in utero, and dramatically improves accessibility to the early fetal stages. Furthermore, in combination with gene-delivery techniques such as electroporation, gene expression can be manipulated in a tissue- and region-specific manner (Osumi and Inoue, 2001 Takahashi et al., 2002). Here we introduce this combinatorial proce dure applying of the electroporation technique to whole-embryo culture system. We then illustrate the use of this approach in the developmental neurobiology by present ing our findings on molecular mechanisms controlling stem cell maintenance and neuronal migration in the embryonic cortex. Finally, we will discuss future directions and a potential widespread value of this method in developmental biology. © 2009 Springer Japan.

To produce precise number of neurons and glial cells from neuroepithelial cells, the progeression and exit of the cell cycle should accurately be coordinated. In mammalian neuroepithelial cells, the molecular and cellular mechanisms coordinating the cell cycle progression and neuronal differentiation is yet unknown. Recently, we noticed that a certain cell cycle regulator protein localized in the basal endfeet of the mouse neuroepithelial cells. However, it is difficult to know in which cell cycle phase the protein localizes, because suitable cell cycle markers expressed at the endfeet of the neuroepithelial cells are missing. To address this issue, we performed sequential labeling of neuroepithelial cells by introducing EGFP-F cDNA into cultured mouse embryos using electroporation and short pulse labeling of S-phase cells by incorporating BrdU, and analyzed the cell cycle phase of EGFP-F labeled cells by using antibodies to BrdU and PH3 (a M-phase marker). We found that EGFP-F-labled cells were in S-phase to G1-phase, but not in G2-phase to M-phase 6 hours after electroporation, and that both of the nucleus and the whole outline of neuroepithelial cell including bipolar processes were clearly visualized. These results suggest that our labeling strategy makes it possible to characterize the cell cycle of neuroepithelial cells, and therefore the cell cycle-dependent distribution of cytoplasmic proteins at the endfeet would be elucidated in the future.

The vertebrate nervous system consists of a huge number of neurons and glial cells. These cell types are generated from neuroepithelial cells at precise positions during development. However, little is known about how kinetics of neuroepithelial cells is regulated, and how the balance of proliferation and differentiation are genetically coordinated. To elucidate cellular mechanisms underlying such complex cell behaviors in the neuroepithelium, we established time-lapse imaging system by applying confocal laser-scanning microscopy for the rat spinal cord slice culture. By electroporation the spinal cord with expression plasmids of fluorescent proteins, nuclear movement of neuroepithelial cells and morphological change on their radial fibers were clearly visualized. Four-dimensional (4-D) time-lapse analyses allowed us to investigate the spatiotemporal dynamics of neuroepithelial cells. These approaches make it possible to analyze functions of genes that regulate cell kinetics and cell morphology.