Annual Microscopy and MicroAnalysis Meeting,
5 to 9 August 2001
Long Beach, California


 

MAPPING ORGANISM EXPRESSION LEVELS AT CELLULAR RESOLUTION
IN DEVELOPING DROSOPHILA

David W. Knowles*, Mark D. Biggin+, Stephen Richards+, Damir Sudar*

Departments of Cell and Molecular Biology* and Genome Sciences+,
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Sequence specific transcription factors are the predominant regulators of animal gene expression controlling nearly all biological processes. We are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within an entire organism. Pregastrula Drosophila embryos have been chosen because these embryos allow high resolution 3D optical imaging since they comprise a single layer of dividing cells surrounding a yolk sac. In addition, the transcription network controlling gene expression is well characterized in early Drosophila embryos[1], and is being further dissected by a multi-laboratory collaboration,  the Berkeley Collaboration in Drosophila Genomics, which encompasses this work.

Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy (Figure 1). Intensity-based segmentation of the total DNA image[2] produces a nuclear mask which defines the nuclear boundaries, their location and the number of cells within the embryo (Figure 2). Presently, dilation of the nuclear volumes into their nearest-neighbours[3] is used to estimate the boundary of the cell (Figure 3) and superposition of these images produces a morphological mask defining each cell and its nucleus. For every stage of developement an "average" morphology is chosen as a reference "atlas" to which images from different embryo are compared. The morphological mask is then used to segment both the total-DNA and gene-product images. Average total DNA brightness per optical slice decreases as optical penetration increases into the embryo and this dependence is used to normalize the brightness of the gene-product images. What results is a map of the relative amount of the specific gene product expressed within the cytoplasm and nucleus of every cell in the embryo, and this in turn is compared to the morphological atlas of that stage.

Our goal is to develop a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. This database will be combined with others that describe additional key parameters of transcriptional control, such as the DNA binding specificities of transcription factors. With this information, we hope to uncover the rules determining how patterns of gene expression are generated.

REFERENCES
[1] Mark D. Biggin, Robert Tjian 2001
Transcriptional regulation in Drosophila: the post-genome challenge
Functional Integrative Genomics in-press

[2] Ortiz de Solórzano C.,et al 1999
Segmentation of Confocal Microscope Images of Cell Nuclei in Thick Tissue Sections
Journal of Microscopy, 193(3):212-226

[3] Knowles D.W., et al. 2000
Analysis of the 3D spatial organization of cells and sub cellular structures in tissue
In Optical Diagnostics of Living Cells III, Daniel L. Farkas, Robert C. Leif Editors
Proceedings of SPIE. Vol 3921:66-73