ABSTRACT
Transcription is episodic, consisting of a series of discontinuous bursts. Using live-imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.
Subject(s)
Chromosomes/metabolism , Drosophila melanogaster/metabolism , Enhancer Elements, Genetic , Transcription, Genetic , Transcriptional Activation , Animals , Drosophila melanogaster/genetics , Embryo, Nonmammalian/metabolism , Female , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Insulator Elements , Male , Promoter Regions, GeneticABSTRACT
Although fluctuations in transcription factor (TF) dosage are often well tolerated, TF dosage modulation can change the target gene expression dynamics and result in significant non-lethal developmental phenotypes. Using MS2/MCP-mediated quantitative live imaging in early Drosophila embryos, we analyzed how changing levels of the gap gene Krüppel (Kr) affects transcriptional dynamics of the pair-rule gene even-skipped (eve). Halving the Kr dosage leads to a transient posterior expansion of the eve stripe 2 and an anterior shift of stripe 5. Surprisingly, the most significant changes are observed in eve stripes 3 and 4, the enhancers of which do not contain Kr-binding sites. In Kr heterozygous embryos, both stripes 3 and 4 display narrower widths, anteriorly shifted boundaries and reduced mRNA production levels. We show that Kr dosage indirectly affects stripe 3 and 4 dynamics by modulating other gap gene dynamics. We quantitatively correlate moderate body segment phenotypes of Kr heterozygotes with spatiotemporal changes in eve expression. Our results indicate that nonlinear relationships between TF dosage and phenotypes underlie direct TF-DNA and indirect TF-TF interactions.
Subject(s)
Drosophila Proteins , Homeodomain Proteins , Kruppel-Like Transcription Factors , Animals , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Kruppel-Like Transcription Factors/metabolismABSTRACT
The transcription of DNA by RNA polymerase occurs as a discontinuous process described as transcriptional bursting. This bursting behavior is observed across species and has been quantified using various stochastic modeling approaches. There is a large body of evidence that suggests the bursts are actively modulated by transcriptional machinery and play a role in regulating developmental processes. Under a commonly used two-state model of transcription, various enhancer-, promoter- and chromatin microenvironment-associated features are found to differentially influence the size and frequency of bursting events - key parameters of the two-state model. Advancement of modeling and analysis tools has revealed that the simple two-state model and associated parameters may not sufficiently characterize the complex relationship between these features. The majority of experimental and modeling findings support the view of bursting as an evolutionarily conserved transcriptional control feature rather than an unintended byproduct of the transcription process. Stochastic transcriptional patterns contribute to enhanced cellular fitness and execution of proper development programs, which posit this mode of transcription as an important feature in developmental gene regulation. In this Review, we present compelling examples of the role of transcriptional bursting in development and explore the question of how stochastic transcription leads to deterministic organism development.
Subject(s)
Gene Expression Regulation , Transcription, Genetic , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Chromatin/genetics , Chromosomes/metabolism , Transcriptional ActivationABSTRACT
How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes incis and intrans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.
Subject(s)
Drosophila melanogaster/genetics , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Transcriptional Activation , Animals , Animals, Genetically Modified , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Genes, Reporter , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.
Subject(s)
Cell Communication/physiology , Optogenetics , Phosphatidylinositol 3-Kinases/metabolism , ras Proteins/metabolism , Animals , Cell Line , Drosophila , Embryo, Nonmammalian , Mice , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction , Temperature , Zebrafish , ras Proteins/geneticsABSTRACT
Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.
Subject(s)
Drosophila/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Animals , Cell Cycle , Drosophila/metabolism , Ploidies , RNA, Messenger/metabolism , Zygote/metabolismABSTRACT
Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail.
Subject(s)
Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Mitosis/physiology , Snail Family Transcription Factors/metabolism , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Embryo, Nonmammalian , Enhancer Elements, Genetic/genetics , Mitosis/genetics , Snail Family Transcription Factors/genetics , Transgenes/geneticsABSTRACT
The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, we measured the contribution of individual binding sites in transcriptional regulation. We show that binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, we show that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. We propose that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.
Subject(s)
Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Enhancer Elements, Genetic , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Female , Gene Expression Regulation, Developmental , Male , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Spatio-Temporal AnalysisABSTRACT
Proper enhancer-promoter interactions are essential to maintaining specific transcriptional patterns and preventing ectopic gene expression. Drosophila is an ideal model organism to study transcriptional regulation due to extensively characterized regulatory regions and the ease of implementing new genetic and molecular techniques for quantitative analysis. The mechanisms of enhancer-promoter interactions have been investigated over a range of length scales. At a DNA level, compositions of both enhancer and promoter sequences affect transcriptional dynamics, including duration, amplitude, and frequency of transcriptional bursting. 3D chromatin topology is also important for proper enhancer-promoter contacts. By working competitively or cooperatively with one another, multiple, simultaneous enhancer-enhancer, enhancer-promoter, and promoter-promoter interactions often occur to maintain appropriate levels of mRNAs. For some long-range enhancer-promoter interactions, extra regulatory elements like insulators and tethering elements are required to promote proper interactions while blocking aberrant ones. This review provides an overview of our current understanding of the mechanism of enhancer-promoter interactions and how perturbations of such interactions affect transcription and subsequent physiological outcomes.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Enhancer Elements, Genetic , Promoter Regions, Genetic , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Proper gene control across space and time is crucial for the seamless execution of various cellular functions. Rapid advancements in genome-wide studies revealed that in addition to genetic mutations, epigenetic modifications also play an important role in cellular processes and disease development. Epigenetic modifications, including DNA methylation and post-translational modifications on histones via methylation, acetylation, phosphorylation, etc., do not alter DNA sequences. Yet, disruptions of the epigenome can still induce gene malfunction, aberrant cell differentiation, proliferation, and apoptosis, resulting in various diseases such as cancer, neurological disorders, and autoimmune diseases. This review describes the association between epigenetic modifications and disease phenotypes, current techniques to perturb epigenome and analyze changes in gene expression, and perspectives on future epigenetic research.
ABSTRACT
Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. Here, we present evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. We observe sequential anterior shifting of the stripes along the anterior to posterior axis, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila/embryology , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Transcription Factors/genetics , Animals , Drosophila/genetics , Embryo, Nonmammalian/physiology , Enhancer Elements, Genetic/physiology , Gene Editing , Gene Regulatory NetworksABSTRACT
The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Embryo, Nonmammalian/metabolism , Phosphoproteins/metabolism , Alleles , Animals , Calpain/genetics , Calpain/metabolism , DNA-Binding Proteins/genetics , Drosophila/genetics , Drosophila Proteins/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Progress of development is commonly reconstructed from imaging snapshots of chemical or mechanical processes in fixed tissues. As a first step in these reconstructions, snapshots must be spatially registered and ordered in time. Currently, image registration and ordering are often done manually, requiring a significant amount of expertise with a specific system. However, as the sizes of imaging data sets grow, these tasks become increasingly difficult, especially when the images are noisy and the developmental changes being examined are subtle. To address these challenges, we present an automated approach to simultaneously register and temporally order imaging data sets. The approach is based on vector diffusion maps, a manifold learning technique that does not require a priori knowledge of image features or a parametric model of the developmental dynamics. We illustrate this approach by registering and ordering data from imaging studies of pattern formation and morphogenesis in three model systems. We also provide software to aid in the application of our methodology to other experimental data sets.
Subject(s)
Computational Biology/methods , Datasets as Topic , Embryonic Development/physiology , Image Processing, Computer-Assisted/methods , Software , Animals , Artificial Intelligence , Drosophila/embryology , Zebrafish/physiologyABSTRACT
Dynamical processes in biology are studied using an ever-increasing number of techniques, each of which brings out unique features of the system. One of the current challenges is to develop systematic approaches for fusing heterogeneous datasets into an integrated view of multivariable dynamics. We demonstrate that heterogeneous data fusion can be successfully implemented within a semi-supervised learning framework that exploits the intrinsic geometry of high-dimensional datasets. We illustrate our approach using a dataset from studies of pattern formation in Drosophila. The result is a continuous trajectory that reveals the joint dynamics of gene expression, subcellular protein localization, protein phosphorylation, and tissue morphogenesis. Our approach can be readily adapted to other imaging modalities and forms a starting point for further steps of data analytics and modeling of biological dynamics.
Subject(s)
Body Patterning/physiology , Image Processing, Computer-Assisted/methods , Models, Biological , Animals , Computational Biology , Drosophila/growth & development , Microscopy, Confocal , Supervised Machine LearningABSTRACT
Developmental biology has traditionally relied on qualitative analyses; recently, however, as in other fields of biology, researchers have become increasingly interested in acquiring quantitative knowledge about embryogenesis. Advances in fluorescence microscopy are enabling high-content imaging in live specimens. At the same time, microfluidics and automation technologies are increasing experimental throughput for studies of multicellular models of development. Furthermore, computer vision methods for processing and analyzing bioimage data are now leading the way toward quantitative biology. Here, we review advances in the areas of fluorescence microscopy, microfluidics, and data analysis that are instrumental to performing high-content, high-throughput studies in biology and specifically in development. We discuss a case study of how these techniques have allowed quantitative analysis and modeling of pattern formation in the Drosophila embryo.
Subject(s)
High-Throughput Screening Assays/methods , Image Processing, Computer-Assisted/methods , Microfluidic Analytical Techniques/methods , Microfluidics/methods , Microscopy, Fluorescence/methods , Morphogenesis/physiology , Animals , HumansABSTRACT
ERK controls gene expression in development, but mechanisms that link ERK activation to changes in transcription are not well understood. We used high-resolution analysis of signaling dynamics to study transcriptional interpretation of ERK signaling during Drosophila embryogenesis, at a stage when ERK induces transcription of intermediate neuroblasts defective (ind), a gene essential for patterning of the nerve cord. ERK induces ind by antagonizing its repression by Capicua (Cic), a transcription factor that acts as a sensor of receptor tyrosine kinases in animal development and human diseases. A recent study established that active ERK reduces the nuclear levels of Cic, but it remained unclear whether this is required for the induction of Cic target genes. We provide evidence that Cic binding sites within the regulatory DNA of ind control the spatial extent and the timing of ind expression. At the same time, we demonstrate that ERK induces ind before Cic levels in the nucleus are reduced. Based on this, we propose that ERK-dependent relief of gene repression by Cic is a two-step process, in which fast reduction of repressor activity is followed by slower changes in nuclear localization and overall protein levels. This may be a common feature of systems in which ERK induces genes by relief of transcriptional repression.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental/physiology , HMGB Proteins/metabolism , Homeodomain Proteins/metabolism , MAP Kinase Signaling System/physiology , Repressor Proteins/metabolism , Animals , Animals, Genetically Modified , Body Patterning/genetics , Body Patterning/physiology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Green Fluorescent Proteins/genetics , HMGB Proteins/genetics , Homeodomain Proteins/genetics , Humans , MAP Kinase Signaling System/genetics , Microfluidics , Phosphorylation/physiology , Repressor Proteins/geneticsABSTRACT
The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Transcription Factors/metabolism , Animals , Animals, Genetically Modified , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Body Patterning/genetics , Body Patterning/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Feedback, Physiological , Gene Expression Regulation, Developmental , Genes, Insect , HMGB Proteins/genetics , HMGB Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Models, Biological , Mutation , Nuclear Proteins/genetics , Phosphoproteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Invertebrate Peptide/genetics , Receptors, Invertebrate Peptide/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction , Transcription Factors/geneticsABSTRACT
Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.
Subject(s)
Embryonic Development , Gene Expression Regulation, Developmental , Animals , Embryonic Development/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Embryo, Nonmammalian/metabolism , Drosophila/embryology , Drosophila/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Transcription, Genetic , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to quantitatively measure the rate of RNA polymerase II elongation for each actively transcribing nucleus in living Drosophila embryos. The elongation rate is calculated by measuring the effective distance and the time elapsed between MS2 and PP7 trajectories. We describe steps for preparing embryo samples, performing live imaging, and measuring the elongation rate. For complete details on the use and execution of this protocol, please refer to Keller et al.1.
Subject(s)
Embryo, Nonmammalian , RNA Polymerase II , Animals , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Embryo, Nonmammalian/metabolism , Drosophila/embryology , Drosophila/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/geneticsABSTRACT
Studies on the dynamics of single cell phenotyping have been hampered by the lack of quantitative high-throughput metabolism assays. Extracellular acidification, a prominent phenotype, yields significant insights into cellular metabolism, including tumorigenicity. Here, we develop a versatile microfluidic system for single cell optical pH analysis (SCO-pH), which compartmentalizes single cells in 140-pL droplets and immobilizes approximately 40,000 droplets in a two-dimensional array for temporal extracellular pH analysis. SCO-pH distinguishes cells undergoing hyperglycolysis induced by oligomycin A from untreated cells by monitoring their extracellular acidification. To facilitate pH sensing in each droplet, we encapsulate a cell-impermeable pH probe whose fluorescence intensities are quantified. Using this approach, we can differentiate hyperglycolytic cells and concurrently observe single cell heterogeneity in extracellular acidification dynamics. This high-throughput system will be useful in applications that require dynamic phenotyping of single cells with significant heterogeneity.