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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.

Developmental enhancers are DNA sequences that when bound to transcription factors dictate specific patterns of gene expression during development. It has been proposed that the evolution of such cis-regulatory elements is a major source of adaptive evolution; however, the regulatory and evolutionary potential of such elements remains little understood, masked by selective constraints, drift and contingency. Here, using mutation libraries inDrosophila melanogasterembryos, we observed that most mutations in classical developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native cell- and tissue-types. In contrast, random sequences often acted as developmental enhancers, driving expression across a range of levels and cell-types, in patterns consistent with transcription factor motifs therein; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently and resulting in higher levels of expression. Together, our findings suggest that the adaptive phenotypic landscapes of developmental enhancers are constrained by both enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity ofde novoelements is a primary source of phenotypic novelty.QUOTE“Chance and chance alone has a message for us.” Milan Kundera,The Unbearable Lightness of Being

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.

Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. Here, to explore the evolutionary capacity of promoter variation, we surveyed an unbiased mutation library for three promoters in Drosophila melanogaster. We found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers.

Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. Here, to explore the evolutionary capacity of promoter variation, we surveyed an unbiased mutation library for three promoters in Drosophila melanogaster . We found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers. This article is part of the theme issue ‘Interdisciplinary approaches to predicting evolutionary biology’.

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