ResearchPad - tools-and-resources https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Cre-assisted fine-mapping of neural circuits using orthogonal split inteins]]> https://www.researchpad.co/article/elastic_article_7268 In humans – as well as flies and most other animals – the brain controls how we move and behave, and regulates heartbeat, breathing and other core processes. To perform these different roles, cells known as neurons form large networks that quickly carry messages around the brain and to other parts of the body. In order to fully understand how the brain works, it is important to first understand how individual neurons connect to each other and operate within these networks.

Fruit flies and other animals with small brains are often used as models to study how the brain works. There are several methods currently available that allow researchers to manipulate small groups of fruit fly neurons for study, and in some cases it is even possible to target individual neurons. However, it remains an aspirational goal to be able to target every neuron in the fly brain individually.

The Gal4-UAS system is a way of manipulating gene activity widely used to study neurons in fruit flies. The system consists of two parts: a protein that can bind DNA and control the activity of genes (Gal4); and a genetic sequence (the UAS) that tells Gal4 where to bind and therefore which genes to activate. Fruit flies can be genetically engineered so that only specific cells make Gal4. This makes it possible, for example, to limit the activity of a gene under the control of the UAS to a specific set of neurons and therefore to identify or target these neurons. Luan et al. developed a new technique named SpaRCLIn that allows the targeting of a subset of neurons within a group already identified with the Gal4-UAS system.

During embryonic development, all neurons originate from a small pool of cells called neuroblasts, and it is possible to target the descendants of particular neuroblasts. SpaRCLIn exploits this strategy to limit the activity of Gal4 to smaller and smaller numbers of neuroblast descendants. In this way, Luan et al. found that SpaRCLIn was routinely capable of limiting patterns of Gal4 activity to one, or a few, neurons at a time. Further experiments used SpaRCLIn to identify two pairs of neurons that trigger a well-known feeding behavior in fruit flies. Luan et al. also developed a SpaRCLIn toolkit that will form the basis of a community resource other researchers can use to study neurons in fruit flies. These findings could also benefit researchers developing similar tools in mice and other animals.

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<![CDATA[Top-down machine learning approach for high-throughput single-molecule analysis]]> https://www.researchpad.co/article/N957aad02-2c00-4587-a7f5-2b73aea07b8d During a chemical or biological process, a molecule may transition through a series of states, many of which are rare or short-lived. Advances in technology have made it easier to detect these states by gathering large amounts of data on individual molecules. However, the increasing size of these datasets has put a strain on the algorithms and software used to identify different molecular states.

Now, White et al. have developed a new algorithm called DISC which overcomes this technical limitation. Unlike most other algorithms, DISC requires minimal input from the user and uses a new method to group the data into categories that represent distinct molecular states. Although this new approach produces a similar end-result, it reaches this conclusion much faster than more commonly used algorithms.

To test the effectiveness of the algorithm, White et al. studied how individual molecules of a chemical known as cAMP bind to parts of proteins called cyclic nucleotide binding domains (or CNDBs for short). A fluorescent tag was attached to single molecules of cAMP and data were collected on the behavior of each molecule. Previous evidence suggested that when four CNDBs join together to form a so-called tetramer complex, this affects the binding of cAMP. Using the DISC system, White et al. showed that individual cAMP molecules interact with all four domains in a similar way, suggesting that the binding of cAMP is not impacted by the formation of a tetramer complex.

Analyzing this data took DISC less than 20 minutes compared to existing algorithms which took anywhere between four hours and two weeks to complete. The enhanced speed of the DISC algorithm could make it easier to analyze much larger datasets from other techniques in addition to fluorescence. This means that a greater number of states can be sampled, providing a deeper insight into the inner workings of biological and chemical processes.

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<![CDATA[A calibrated optogenetic toolbox of stable zebrafish opsin lines]]> https://www.researchpad.co/article/N1ccb8a53-68b9-48c2-94c3-754ddf9cf911

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

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<![CDATA[A synthetic dataset primer for the biobehavioural sciences to promote reproducibility and hypothesis generation]]> https://www.researchpad.co/article/Nc154045a-ca65-4a9a-bdc2-105149669d9e

Open research data provide considerable scientific, societal, and economic benefits. However, disclosure risks can sometimes limit the sharing of open data, especially in datasets that include sensitive details or information from individuals with rare disorders. This article introduces the concept of synthetic datasets, which is an emerging method originally developed to permit the sharing of confidential census data. Synthetic datasets mimic real datasets by preserving their statistical properties and the relationships between variables. Importantly, this method also reduces disclosure risk to essentially nil as no record in the synthetic dataset represents a real individual. This practical guide with accompanying R script enables biobehavioural researchers to create synthetic datasets and assess their utility via the synthpop R package. By sharing synthetic datasets that mimic original datasets that could not otherwise be made open, researchers can ensure the reproducibility of their results and facilitate data exploration while maintaining participant privacy.

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<![CDATA[Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity]]> https://www.researchpad.co/article/N9f8a4863-a649-4758-a829-2914bd3c5856

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

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<![CDATA[A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC]]> https://www.researchpad.co/article/Na61a0af3-6d95-4951-aa97-790d4d9fb098

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

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<![CDATA[Proteome profile of peripheral myelin in healthy mice and in a neuropathy model]]> https://www.researchpad.co/article/N27b073a3-f881-43d8-adae-f8ad384659e0

Proteome and transcriptome analyses aim at comprehending the molecular profiles of the brain, its cell-types and subcellular compartments including myelin. Despite the relevance of the peripheral nervous system for normal sensory and motor capabilities, analogous approaches to peripheral nerves and peripheral myelin have fallen behind evolving technical standards. Here we assess the peripheral myelin proteome by gel-free, label-free mass-spectrometry for deep quantitative coverage. Integration with RNA-Sequencing-based developmental mRNA-abundance profiles and neuropathy disease genes illustrates the utility of this resource. Notably, the periaxin-deficient mouse model of the neuropathy Charcot-Marie-Tooth 4F displays a highly pathological myelin proteome profile, exemplified by the discovery of reduced levels of the monocarboxylate transporter MCT1/SLC16A1 as a novel facet of the neuropathology. This work provides the most comprehensive proteome resource thus far to approach development, function and pathology of peripheral myelin, and a straightforward, accurate and sensitive workflow to address myelin diversity in health and disease.

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<![CDATA[Regulation of mRNA translation by a photoriboswitch]]> https://www.researchpad.co/article/Nc2ffddfd-1feb-4795-aff8-54d3618d464d

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)–a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.

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<![CDATA[Transcriptional landscape of myogenesis from human pluripotent stem cells reveals a key role of TWIST1 in maintenance of skeletal muscle progenitors]]> https://www.researchpad.co/article/N593d86a6-5262-4dc2-a035-df11c396a68e

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).

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<![CDATA[NINscope, a versatile miniscope for multi-region circuit investigations]]> https://www.researchpad.co/article/N6ccaf3a9-c5e3-445b-baf1-1f7ec71521d7

Miniaturized fluorescence microscopes (miniscopes) have been instrumental to monitor neural signals during unrestrained behavior and their open-source versions have made them affordable. Often, the footprint and weight of open-source miniscopes is sacrificed for added functionality. Here, we present NINscope: a light-weight miniscope with a small footprint that integrates a high-sensitivity image sensor, an inertial measurement unit and an LED driver for an external optogenetic probe. We use it to perform the first concurrent cellular resolution recordings from cerebellum and cerebral cortex in unrestrained mice, demonstrate its optogenetic stimulation capabilities to examine cerebello-cerebral or cortico-striatal connectivity, and replicate findings of action encoding in dorsal striatum. In combination with cross-platform acquisition and control software, our miniscope is a versatile addition to the expanding tool chest of open-source miniscopes that will increase access to multi-region circuit investigations during unrestrained behavior.

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<![CDATA[Free circular introns with an unusual branchpoint in neuronal projections]]> https://www.researchpad.co/article/N311d8778-192f-4d2c-82c8-48193bc3b93e

The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

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<![CDATA[Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology]]> https://www.researchpad.co/article/N5a1b686e-52a6-4bec-8ec9-0dfa740cbf40 Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter ‘zoospores’. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes. ]]> <![CDATA[GLO-Roots: an imaging platform enabling multidimensional characterization of soil-grown root systems]]> https://www.researchpad.co/article/5989da7cab0ee8fa60b98b39

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multi-dimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated-imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression, and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes.

DOI: http://dx.doi.org/10.7554/eLife.07597.001

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<![CDATA[Automatic discovery of cell types and microcircuitry from neural connectomics]]> https://www.researchpad.co/article/5989da87ab0ee8fa60b9ca39

Neural connectomics has begun producing massive amounts of data, necessitating new analysis methods to discover the biological and computational structure. It has long been assumed that discovering neuron types and their relation to microcircuitry is crucial to understanding neural function. Here we developed a non-parametric Bayesian technique that identifies neuron types and microcircuitry patterns in connectomics data. It combines the information traditionally used by biologists in a principled and probabilistically coherent manner, including connectivity, cell body location, and the spatial distribution of synapses. We show that the approach recovers known neuron types in the retina and enables predictions of connectivity, better than simpler algorithms. It also can reveal interesting structure in the nervous system of Caenorhabditis elegans and an old man-made microprocessor. Our approach extracts structural meaning from connectomics, enabling new approaches of automatically deriving anatomical insights from these emerging datasets.

DOI: http://dx.doi.org/10.7554/eLife.04250.001

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<![CDATA[Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf462

The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.

DOI: http://dx.doi.org/10.7554/eLife.24463.001

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<![CDATA[A robust activity marking system for exploring active neuronal ensembles]]> https://www.researchpad.co/article/5989daf0ab0ee8fa60bc0d3b

Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system’s versatility.

DOI: http://dx.doi.org/10.7554/eLife.13918.001

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<![CDATA[Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses]]> https://www.researchpad.co/article/5989d9d1ab0ee8fa60b64538

The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and super-resolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed sub-synaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers.

DOI: http://dx.doi.org/10.7554/eLife.20560.001

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<![CDATA[Peripheral optogenetic stimulation induces whisker movement and sensory perception in head-fixed mice]]> https://www.researchpad.co/article/5989da77ab0ee8fa60b96fa4

We discovered that optical stimulation of the mystacial pad in Emx1-Cre;Ai27D transgenic mice induces whisker movements due to activation of ChR2 expressed in muscles controlling retraction and protraction. Using high-speed videography in anesthetized mice, we characterize the amplitude of whisker protractions evoked by varying the intensity, duration, and frequency of optogenetic stimulation. Recordings from primary somatosensory cortex (S1) in anesthetized mice indicated that optogenetic whisker pad stimulation evokes robust yet longer latency responses than mechanical whisker stimulation. In head-fixed mice trained to report optogenetic whisker pad stimulation, psychometric curves showed similar dependence on stimulus duration as evoked whisker movements and S1 activity. Furthermore, optogenetic stimulation of S1 in expert mice was sufficient to substitute for peripheral stimulation. We conclude that whisker protractions evoked by optogenetic activation of whisker pad muscles results in cortical activity and sensory perception, consistent with the coding of evoked whisker movements by reafferent sensory input.

DOI: http://dx.doi.org/10.7554/eLife.14140.001

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<![CDATA[Selections that isolate recombinant mitochondrial genomes in animals]]> https://www.researchpad.co/article/5989da9aab0ee8fa60ba3535

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

DOI: http://dx.doi.org/10.7554/eLife.07247.001

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<![CDATA[High-throughput mathematical analysis identifies Turing networks for patterning with equally diffusing signals]]> https://www.researchpad.co/article/5989da67ab0ee8fa60b921a9

The Turing reaction-diffusion model explains how identical cells can self-organize to form spatial patterns. It has been suggested that extracellular signaling molecules with different diffusion coefficients underlie this model, but the contribution of cell-autonomous signaling components is largely unknown. We developed an automated mathematical analysis to derive a catalog of realistic Turing networks. This analysis reveals that in the presence of cell-autonomous factors, networks can form a pattern with equally diffusing signals and even for any combination of diffusion coefficients. We provide a software (available at http://www.RDNets.com) to explore these networks and to constrain topologies with qualitative and quantitative experimental data. We use the software to examine the self-organizing networks that control embryonic axis specification and digit patterning. Finally, we demonstrate how existing synthetic circuits can be extended with additional feedbacks to form Turing reaction-diffusion systems. Our study offers a new theoretical framework to understand multicellular pattern formation and enables the wide-spread use of mathematical biology to engineer synthetic patterning systems.

DOI: http://dx.doi.org/10.7554/eLife.14022.001

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