Exploiting Analysis of Heterogeneity to Increase the Information Content Extracted from Fluorescence Micrographs of...

Zebrafish embryos are a near-ideal animal model for drug discovery because of their high genetic and physiological similarity to mammals, small size, high fecundity, and optical transparency. The latter properties make zebrafish at larval stages especially suited for high-content analysis and high throughput screening (HTS). However, inherent biological complexity and the inability to screen multiple specimens in a single well present a challenge for HTS because limiting replicates and high variability often prevent assays from reaching the stringent performance criteria demanded of large-scale screening assays. In this report, we present methodology that overcomes these obstacles. We used our previously developed Tg(lhx1a:EGFP)^pt303 line, which expresses a fluorescent transgene that enables live real-time measurements of kidney progenitor cell expansion. Since transgenes are expressed in specific cell populations, whose localization is precisely controlled, both spatially and temporally, we considered the developing embryo to be a "host" for a cell population, analogous to a well of a cell culture microplate, rather than a single specimen. By adopting this view, parameters routinely used to analyze cultured cells became applicable to characterize and quantify zebrafish transgene appearance beyond the overall intensity or area measurements, which are analogous to calculating well average data. Using the pixel-level distribution of transgene intensity as a proxy to cell-level data, we applied population-based intensity and heterogeneity measurements to quantitatively describe and characterize transgene expression in each embryo. Subsequent linear discriminant analysis on eight such parameters captured and condensed this information into a single assay parameter that maximizes the difference between positive and negative responses. The improvements in assay performance resulted in the Tg(lhx1a:EGFP)^pt303 assay achieving HTS compatible assay performance in multi-day variability studies, documenting readiness for HTS of compounds that expand kidney progenitor cell populations.