Title:
Multi-dimensional, ‘minimum-damage’ live-cell imaging for mouse preimplantation development and its application for the embryo assessment.

Kazuo Yamagata, Rinako Suetsugu, Eiji Mizutani, and Teruhiko Wakayama
Riken, Center for Developmental Biology, Laboratory for Genomic Reprogramming

Abstract:
Mammalian fertilization and subsequent preimplantation development are irreversible processes of the dynamic conversion of two highly specialized cells?the sperm and egg?into totipotent zygotes. They are achieved by the tightly coordinated regulation of a great variety of temporal and spatial changes. Especially, the nuclear events, i.e., changes in nuclear and chromosome architecture and global changes in epigenetic status, take place and these are thought to be associated with de-differentiation and differentiation of embryo, so-called nuclear reprogramming process. Therefore, three-dimensional live-cell imaging technique will be valuable to analyze these events at a molecular level. Moreover, a system enabling the embryos to develop to term normally after the imaging would allow both retrospective and prospective links between the phenomena observed in early stages of embryogenesis and the further developmental potential. Here, we developed a live-cell imaging method based on the expression of fluorescent proteins using mRNA injection and time-lapse florescence microscopy. This system enabled six-dimensional live-cell imaging of mouse preimplantation development ( x , y and z axes, time-lapse, multicolor and multisample). Importantly, by optimizing the conditions for imaging, the procedure itself was not detrimental, ‘minimum-damage’ to full-term development, although it is a prolonged imaging process.
Then, by using our imaging technique, we assessed ‘ill-fated’ embryos such as intracytoplasmic sperm injection (ICSI)-generated and cloned embryos by focusing on nuclear dynamics and chromosome integrity. One major finding was that abnormal chromosome segregation frequently occurred during mitotic divisions at early cleavage stage, and this was a reason for the early gestational loss in these embryos. In this seminar, in addition to imaging of chromosome dynamics, I will introduce other observations such as live-imaging of epigenetic status (DNA methylation and histone modifications) and long-term imaging of ES cell derivation process. I will be happy if you give me some suggestions and discussions about application of our imaging technique.

References:
・ Yamagata K, Suetsugu R and Wakayama T. Long-term, Six-dimensional Live-cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-term Development. J Reprod Dev 2009; 55: 328-331.

・ Yamagata K, Suetsugu R and Wakayama T. Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection. Hum. Reprod. 2009; 24(10):2490-9.

・山縣一夫、 6 章「観察（応用編）に関する Q&A 」、「顕微鏡活用なるほど Q&A 」（宮戸健二、岡部勝編）、 122 － 146 、羊土社、 2009

・山縣一夫、第 3 章 ―1 「受精・初期胚核ダイナミクスと発生・出生不全」、実験医学増刊号「細胞核 ― 遺伝情報制御と疾患」（平岡泰、原田昌彦、木村宏、田代聡　編）、 Vol. 27, No. 17, 159-166 、 2009