(III) The generation of tools and development of methods for in vivo imaging.

Localization of lipid-modified fluorescent proteins in live embryonic stem (ES) cells.

Central to our work investigating the patterning and morphogenesis of the mammalian embryo is a need for observing events as they take place in their native environment. To understand the morphogenetic movements required to build an embryo and the behavior of stem or progenitor cells in vivo, we want to define, catalog and quantify the stereotypical behaviors of cells that we believe are present in wild type contexts and contrast these findings with those occurring in mutants. Optical imaging is an integral component of the approach we are taking to investigate and quantify cell behavior.

We are developing genetically-encoded optical probes for use in gene targeting and transgenesis regimes in ES cells and mice. Fluorescent protein reporters are used to help visualize cells and tissues in embryos. We have developed strains of mice that provide readouts of cell position (by labeling the nucleus) and cell morphology (by labeling the plasma membrane) permitting the generation of 4-dimensional (3D time-lapse) data at a resolution equivalent to routine histology on fixed tissue.

These reagents provide a powerfull toolbox offering the potential to follow and quantify biological processes at high resolution. Furthermore, the continued evolution of fluorescent proteins coupled with advances in optical imaging modalities, make reporter expressing mice essential reagents for the multidimensional analysis and understanding of biological processes.

Live imaging in the mouse embryo: in vivo chromatin dynamics.

We combine this optical imaging approach with optimized ex utero on-stage embryo culture, thus allowing us to perform high-resolution live cell imaging in vivo in mouse embryos. The unique methodologies and reagents we are developing for live imaging ES cells and mice, represent an excellent platform for investigating and quantifying live cell behaviors, and are being incorporated into various ongoing projects where we want to use in vivo imaging to validate hypothesis concerning cell behaviors deregulated in mutants and establish the baseline for our future work.

Future work will focus on establishing methods to: (i) Directly visualize and identify intracellular components in vivo, especially those involved in cell polarity and migration. (ii) Use photoactivation methods for non-invasive fate mapping.