Live and in color revisited

Live cell imaging takes on a new meaning with transgenic mice expressing GFP

By Laura DeFrancesco
Managing Editor, Bioresearch Online

Last year, in these pages, we looked at technology for imaging gene expression in live cells (see Live and in Color: Monitoring Gene Expression in Real Time)

The point of that article was that gene expression is a dynamic and ephemeral process, and hence, to fully understand it, techniques are needed that can monitor rapid and subtle changes in expression. Now, consider development. What could be more dynamic than that? But as Joshua Sanes told conferees at the ASCB Symposium on Cellular Organization at the Synapse, currently we know more about the molecules involved than the process. Now, however, Sanes and his co-workers at the Washington University School of Medicine (St. Louis) have developed a technique that allows them to monitor developmental processes in live animals using transgenic mice expressing GFP. Live and in color.

Making mice glow
With an eye toward imaging synapse formation, Sanes has been investigating the use of GFP and its spectral variants as probes for growing neurons in developing mouse embryos. To do this, he needed two things—a way of expressing the fluorescent probe in specific cell lineages, and fluorescent probes that wouldn't harm the animal or interfere with development. As described in a Neuron paper last year (ref. 1), Sanes has found both. Working with several neuron-specific promoters, Sanes found that one in particular, thy1, which is a member of the immunoglobin superfamily, can be engineered to drive expression only in neuronal cells. Further, working with four different spectral variants of GFP (green, blue, red, and cyan), he found that mouse development and in particular neuromuscular development was unchanged in transgenic mice expressing any of these molecules in their neurons.

Figure 1: Long-term expression of YFP in motor nerve terminals. Repeated imaging of a neuromuscular junction in developing live mice showed that neither prolonged expression or repeated excitation affected development, as seen in these embryos that were images from 8 weeks up to 44 weeks of gestation.

Serendipity strikes again
Sanes created two dozen lines of transgenic mice expressing one or more GFP variant, and remarkably, they all show different and unique staining patterns---this in spite of the fact that for each GFP variant, the same gene construct was used to create the transgenics. This finding has both theoretical and practical ramifications. The various staining patterns observed in the transgenics likely means that the exact locus of GFP insertion affects where and how it is expressed. And practically, the staining of different individual cell lineages provides a wealth of tools for following specific cells or cell types.

Figure 2: Vital staining of mouse neuromuscular junctions in thy-1-YFP transgenic mice. A mouse expressing YFP in the sternomastoid neuromuscular junction was stained with rhodamine-a bungarotoxin to label post-synaptic acetylcholine receptors.

Take-home lessons
The lessons are many even in this early stage of the work.

• Variation in GFP staining seems to be genetic, which is to say that there is greater variation between transgenic lines than within a line.
  
• Thy-1 is definitely running the show, as staining was entirely neuron specific (once the elements that directed expression in lymphoid tissue were removed from the constructs). Those neurons that express Thy-1 showed the highest levels of GFP expression—motor and sensory neurons, for example, while cortical and cerebellar neurons showed minimal staining.
  
• Transgenic lines can be created that express two or more spectral variants, which will provide a nice way of looking at interactions between groups of neurons in vivo.

For more information: Joshua R. Sanes, Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MI 63110. Email: sanesj@pcg.wustl.edu.

References:

1. Feng G., et al., "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron, 28: 41-21, October 2000.