Laboratory for the Physics of Life Thomas Gregor, Professor of Physics, Princeton University

Julien O. Dubuis, Reba Samanta and Thomas Gregor, Molecular Systems Biology 9: 639 (2013).

Abstract
Quantification of gene expression has become a central tool for understanding genetic networks. In many systems, the only viable way to measure protein levels is by immunofluorescence, which is notorious for its limited accuracy. Using the early Drosophila embryo as an example, we show that careful identification and control of experimental error allows for highly accurate gene expression measurements. We generated antibodies in different host species, allowing for simultaneous staining of four Drosophila gap genes in individual embryos. Careful error analysis of hundreds of expression profiles reveals that less than ~20% of the observed embryo-to-embryo fluctuations stem from experimental error. These measurements make it possible to extract not only very accurate mean gene expression profiles but also their naturally occurring fluctuations of biological origin and corresponding cross-correlations. We use this analysis to extract gap gene profile dynamics with ~1 min accuracy. The combination of these new measurements and analysis techniques reveals a two-fold increase in profile reproducibility owing to a collective network dynamics that relays positional accuracy from the maternal gradients to the pair-rule genes.

Synopsis

Precise analysis of systematic errors shows suitability of immunofluorescence protocols to quantify gene expression means, variances, and cross-correlations. Application to Drosophila gap genes enables reconstructing expression level dynamics and the progression of positional accuracy.

• A careful analysis of the contribution of multiple sources of measurement errors shows that <20% of the observed embryo-to-embryo fluctuations stem from experimental error.
• Intensities and slopes of the borders of gap gene expression patterns simultaneously reach a maximum around 15 min before gastrulation in a precisely coordinated fashion, hinting at an intrinsically collective organization of the gap gene network.
• The reproducibility of gap gene expression levels increases two-fold before reaching a maximum when the overall network dynamics peak. At the same time, the positional accuracy of determining cell fates is half an internuclear distance and uniform along the entire embryo length.

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