Summary Transient activation of the highly conserved extracellular signal regulated kinase (ERK) establishes precise patterns of cell fates in developing tissues. Quantitative parameters of these transients are essentially unknown, but a growing number of studies suggest that changes in these parameters can lead to a broad spectrum of developmental abnormalities. We provide a detailed quantitative picture of an ERK-dependent inductive signaling event in the early Drosophila embryo, an experimental system that offers unique opportunities for high-throughput studies of developmental signaling. Our analysis reveals a spatiotemporal pulse of ERK activation that is consistent with a model in which transient production of a short-ranged ligand feeds into a simple signal interpretation system. The pulse of ERK signaling acts as a switch in controlling the expression of the ERK-target gene. The quantitative approach that led to this model, based on the integration of data from fixed embryos and live imaging, can be extended to other developmental systems patterned by transient inductive signals.

Here we describe a protocol for the fabrication and use of a microfluidic device to rapidly orient >700 Drosophila embryos in parallel for end-on imaging. The protocol describes master microfabrication (∼1 d), polydimethylsiloxane molding (few hours), system setup and device operation (few minutes) and imaging (depending on application). Our microfluidics-based approach described here is one of the first to facilitate rapid orientation for end-on imaging, and it is a major breakthrough for quantitative studies on Drosophila embryogenesis. The operating principle of the embryo trap is based on passive hydrodynamics, and it does not require direct manipulation of embryos by the user; biologists following the protocol should be able to repeat these procedures. The compact design and fabrication materials used allow the device to be used with traditional microscopy setups and do not require specialized fixtures. Furthermore, with slight modification, this array can be applied to the handling of other model organisms and oblong objects.

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Understanding the fundamental principles governing embryogenesis is a key goal of developmental biology. Direct observation of embryogenesis via in vivo live imaging is vital to understanding embryogenesis; yet, tedious sample preparation makes it difficult to acquire large-scale imaging data that is often required to overcome experimental and biological noises for quantitative studies. Furthermore, it is often difficult, and sometimes impossible, to incorporate environmental perturbation for understanding developmental responses to external stimuli. To address this issue, we have developed a method for high-throughput imaging of live embryos, delivering precise environmental perturbations, and unbiased data extraction. This platform includes an optimized microfluidic device specifically for live embryos and also for precise perturbations in the microenvironment of the developing embryos. In addition, we developed software for simple, yet accurate, automated segmentation of fluorescent images, and automated data extraction. Using a quantitative assessment we find that embryos develop normally within the microfluidic device. Finally, we show an application of the high-throughput assay for monitoring developmental responses to external stimuli: anoxia-induced developmental arrest in Drosophila embryos. With slight modifications, the method developed in this work can be applied to many other models of development and other stimulus-response behaviors during development.

Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.