Image processing and analysis for quantifying gene expression from early Drosophila embryos

Correlation of quantities of transcriptional activators and repressors with the mRNA output of target genes is a central issue for modeling gene regulation. In multicellular organisms, both spatial and temporal differences in gene expression must be taken into account; this can be achieved by use of in situ hybridization followed by confocal laser scanning microscopy (CLSM). Here we present a method to correlate the protein levels of the short-range repressor Giant with lacZ mRNA produced by reporter genes using images of Drosophila blastoderm embryos taken by CLSM. The image stacks from CLSM are processed using a semiautomatic algorithm to produce correlations between the repressor levels and lacZ mRNA reporter genes. We show that signals derived from CLSM are proportional to actual mRNA levels. Our analysis reveals that a suggested parabolic form of the background fluorescence in confocal images of early Drosophila embryos is evident most prominently in flattened specimens, with intact embryos exhibiting a more linear background. The data extraction described in this paper is primarily conceived for analysis of synthetic reporter genes that are designed to decipher cis-regulatory grammar, but the techniques are generalizable for quantitative analysis of other engineered or endogenous genes in embryos.