While early embryonic cell lineage analysis is routine (1,2,3), tracking small nuclei in the bottom half of >100 cell embryos is not robust and requires considerable time and expertise (4). We hope to exploit green fluorescent protein technology (5) to develop methods for more robust, faster and highly automated lineage analysis. One potential method relies on two key components. First, Melanie Dunn and Geraldine Seydoux provided a histone H1::GFP transgene which permits observation of chromosomes in living embryos. Second, we developed a microscope image acquisition system capable of taking 64 fluorescent optical sections, 10 times per cell cycle, throughout embryogenesis, at light levels commensurate with normal development. Navigation of simultaneously collected histone::GFP fluorescence and Nomarksi DIC 4D data sets reveals that fluorescence lineage analysis is much easier than scoring divisions by Nomarksi optics. The discrimination of individual nuclei, especially in the bottom half of late embryos, is greatly improved by image processing to reduce the effects of out of focus light. We took advantage of the improved image quality to write a program that identifies the number and 3-dimensional position of each nucleus at a given time point. We are now developing software that compares the position of nuclei at subsequent time points in order to detect cell deaths, divisions and migrations. The output from the tracking program will be in two forms: a traditional lineage diagram showing the time and orientation of each embryonic cell division (until movement at 400 min.); and a series of 3-dimensional coordinates over time. The nuclear coordinates can be used to show a color coded animation of embryonic development from any perspective. Finally, an additional technical barrier to automated cell lineage analysis is to identify a set of histone H1 genes that, in composite, labels chromosomes in every embryonic cell. The existing histone H1::GFP fusion (his-24 gene) is not highly expressed until the 28-cell stage, and is not expressed in a small subset of cells, including the germline precursors. We plan to fuse GFP to the remaining histone H1 genes to determine which are expressed in the HIS-24::GFP negative cells. The automated lineage analysis system should greatly facilitate phenotypic analysis of cell fate mutants and the subsequent dissemination of such information in a viewable, database format. 1. Hird and White, JCB 121:1343(1994). 2. Fire, CABIOS 10:443(1994). 3. Thomas et al., Science 273:603(1996). 4. Schnabel et al., Dev. Biol. 184:234(1997). 5. Chalfie et al., Science 263:802(1994).

Affiliation:
- Dept. of Genetics, Washington University, St. Louis, MO 63110.