Cell Cycle Visualization in Development

Atsushi Miyawaki’s lab has developed a series of neat tools for visualizing cell cycle progress.

For zebrafish, the zFucci system consists of two fluorescent proteins, mKO2 and mAG, that are fused to Cdt1 and geminin genes.  Cell cycle- regulated proteolysis of these fusion proteins causes each cell to display orange fluorescence in G₁ phase nuclei and green fluorescence in both the nucleus and cytoplasm of S/G₂/M phase cells.

Video of cell cycle transitions in culture. Click for the video.

The last time I saw Atsushi give a talk, he showed an incredible time lapse video from the zebrafish cleavage stage that I haven’t been able to find online.  However, here is a video from later in development of the zebrafish that is still pretty remarkable.

Development of a zebrafish visualized by zFucci. Click to see the video.

This two component system has been adapted for watching the transition from neural stem cells to differentiated neurons in living mice. The Color-Timer system uses double transgenics with the fluorescent protein KOr fused to nestin and EGFP fused to doublecortin.  In this system, neural stem cells fluoresce orange, while newly differentiated neurons fluoresce green.

The cerebral cortex of an E14.5 double Tg mouse embryo of nestin/KOr was time-lapse imaged. Click for video

Sugiyama, M., Sakaue-Sawano, A., Iimura, T., Fukami, K., Kitaguchi, T., Kawakami, K., Okamoto, H., Higashijima, S., & Miyawaki, A. (2009). Illuminating cell-cycle progression in the developing zebrafish embryo Proceedings of the National Academy of Sciences, 106 (49), 20812-20817 DOI: 10.1073/pnas.0906464106

Kanki, H., Shimabukuro, M., Miyawaki, A., & Okano, H. (2010). “Color Timer” mice: visualization of neuronal differentiation with fluorescent proteins Molecular Brain, 3 (1) DOI: 10.1186/1756-6606-3-5

The Journal of Visualized Experiments

For technically demanding protocols in neuroscience (or any other science) research, a printed protocol is often insufficient to capture all the essentials of a method.  There are usually numerous ‘tricks’ or things that one must pay attention to that are not included in the printed page.  Or, if they are included, they still lack a vivid description. Many techniques require the novice to be taught the technique from a more experienced colleage. Unfortunately, it is not always easy to find someone skilled to be trained from.  Labs which pioneer the techniques have only a limited amount of time and resources available to train outside scientists. How can advanced scientific skills be distributed more broadly and efficiently? A good place to start is the Journal of Visualized Experiments (JoVE). It’s a YouTube for science protocols.

So that's how you do it!

JoVE is a growing collection of video protocols that walk a researcher through the procedure, allowing one to actually see the steps used, rather than just imaging what performing the protocol might be like. Want to know how to glue a live fruit fly to a stick?  Just watch the video! Wonder how to load calcium dyes onto the cortex of a mouse? Just watch the video!  This looks to be a fantastic resource for people that are learning a technique, that want to see other possible ways to do a procedure, or those who are simply curious about what a neuroscientist actually does at work.

I should make one for glutamate imaging!