No abstract

A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.

Gene regulatory changes underlie much of phenotypic evolution. However, the evolutionary potential of regulatory evolution is unknown, because most evidence comes from either natural variation or limited experimental perturbations. Surveying an unbiased mutation library for a developmental enhancer in Drosophila melanogaster using an automated robotics pipeline, we found that most mutations alter gene expression. Our results suggest that regulatory information is distributed throughout most of a developmental enhancer and that parameters of gene expression-levels, location, and state-are convolved. The widespread pleiotropic effects of most mutations and the codependency of outputs may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse drosophilids reveal mainly stasis and apparent biases in the phenotypes influenced by this enhancer. Developmental enhancers may encode a much higher density of regulatory information than has been appreciated previously, which may impose constraints on regulatory evolution.