Quantitative live imaging describes morphogenetic nuclear movements in early Drosophila embryo.
1) Life Sciences and Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA.
2) Computer Science Division, University of California, Berkeley, CA.
3) Institute for Data Analysis and Visualization, University of California, Davis, CA.
4) Computer Science Department, University of Kaiserslautern, Germany.
The Berkeley Drosophila Transcription Network Project (bdtnp.lbl.gov) is conducting a system-wide analysis of the transcription network in the early Drosophila embryo. As part of this multidisciplinary effort, novel imaging, image analysis and visualization methods have been developed to construct the first three-dimensional (3D) atlas of gene expression and morphology in an embryo at cellular resolution. Our aim is to quantify the relative expression of hundreds of genes in wild type embryos and in a series of mutant embryos, and to map these results onto "stereotypical embryos". Multiple-color in-situ hybridizations are used to fluorescently label gene products of interest, and total DNA is counter-stained. High resolution, multi-channel, 3D images are acquired of entire embryos using two-photon excitation. Individual nuclei are isolated from the DNA-stained images using novel, automated segmentation techniques, and the relative gene expression within and around each nucleus is then quantified. Novel techniques have been developed to register data from many images onto a "stereotypical embryo" and create exquisite quantitative visualizations of the data in 3D. One novel observation we have made is the systematic change in nuclear packing densities during stage 5 (interphase cycle 14, up to gastrulation), which can be seen by comparing fixed embryos of different ages. To understand these nuclear movements, we have studied the process in live histone2A-GFP embryos. Individual embryos were imaged from the 13th cleavage cycle to the start of gastrulation in approximately 3 minute intervals. Rapid temporal sequences were important for tracking individual nuclei but restricted the imaging to the portion of the embryo closest to the objective lens. Orientation of the imaged embryo portions were determined by morphological features during gastrulation and egg-length was determined from optical sections taken through the middle of the embryo. These measurements allowed images from multiple embryos to be combined into whole-embryo-maps. The resulting patterns of nuclear packing density and flow-fields correlate with the dorsal-ventral orientation of the embryo and the future location of the ventral furrow. These results, which support our fixed embryo work, show complex 3D nuclear movements prior to gastrulation that will have to be considered to fully understand dynamics in gene expression patterns.
Last modified May 2, 2006.