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Past Issue in 2023
Volume: 13, Issue: 14
Biochemistry
Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining
Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells.Key features• Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy.• Protocol can be applied to any type of cell that is adherent in culture, including primary cells.• Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes.• Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes.Graphical overviewImaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy. Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).
Biological Engineering
Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments
The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua–derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.
Biophysics
Recoil Measurements in Drosophila Embryos: from Mounting to Image Analysis
Tension and force propagation play a central role in tissue morphogenesis, as they enable sub- and supra-cellular shape changes required for the generation of new structures. Force is often generated by the cytoskeleton, which forms complex meshworks that reach cell–cell or cell–extracellular matrix junctions to induce cellular rearrangements. These mechanical properties can be measured through laser microdissection, which concentrates energy in the tissue of interest, disrupting its cytoskeleton. If the tissue is undergoing tension, this cut will induce a recoil in the surrounding regions of the cut. This protocol describes how one can perform laser microdissection experiments and subsequently measure the recoil speed of the sample of interest. While we explain how to carry out these experiments in Drosophila embryos, the recoil calibration and downstream analyses can be applied to other types of preparations. Key features• Allows measuring tension in live Drosophila embryos with a relatively simple approach. • Describes a quick way to mount a high number of embryos.• Includes a segmentation-free recoil quantification that reduces bias and speeds up analysis.Graphical overview
Cancer Biology
Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging
Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader.Graphical overview
Cell Biology
Optogenetic Induction of Pyroptosis, Necroptosis, and Apoptosis in Mammalian Cell Lines
Regulated cell death plays a key role in immunity, development, and homeostasis, but is also associated with a number of pathologies such as autoinflammatory and neurodegenerative diseases and cancer. However, despite the extensive mechanistic research of different cell death modalities, the direct comparison of different forms of cell death and their consequences on the cellular and tissue level remain poorly characterized. Comparative studies are hindered by the mechanistic and kinetic differences between cell death modalities, as well as the inability to selectively induce different cell death programs in an individual cell within cell populations or tissues. In this method, we present a protocol for rapid and specific optogenetic activation of three major types of programmed cell death: apoptosis, necroptosis, and pyroptosis, using light-induced forced oligomerization of their major effector proteins (caspases or kinases).
Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques
Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.
Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging
Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts.Key features• This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments.• Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers.• The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft.Graphical overview
Developmental Biology
Three-color dSTORM Imaging and Analysis of Recombination Foci in Mouse Spread Meiotic Nuclei
During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience. Key features• Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips. • dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei. • Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci. Graphical overview
Immunology
Monitoring Group 2 Innate Lymphoid Cell Biology in Models of Lung Inflammation
Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4+ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify Lineage−Thy1.2+ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology.Key features• In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33.• Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.• During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels of Gata-3 and Ki-67 and produce greater amounts of IL-5 and IL-13.Graphical overview
LiverQuant: An Improved Method for Quantitative Analysis of Liver Pathology
Current means to quantify cells, gene expression, and fibrosis of liver histological slides are not standardized in the research community and typically rely upon data acquired from a selection of random regions identified in each slide. As such, analyses are subject to selection bias as well as limited subsets of available data elements throughout the slide. A whole-slide analysis of cells and fibrosis would provide for a more accurate and complete quantitative analysis, along with minimization of intra- and inter-experimental variables. Herein, we present LiverQuant, a method for quantifying whole-slide scans of digitized histologic images to render a more comprehensive analysis of presented data elements. After loading images and preparing the project in the QuPath program, researchers are provided with one to two scripts per analysis that generate an average intensity threshold for their staining, automated tissue annotation, and downstream detection of their anticipated cellular matrices. When compared with two standard methodologies for histological quantification, LiverQuant had two significant advantages: increased speed and a 50-fold greater tissue area coverage. Using publicly available open-source code (GitHub), LiverQuant improves the reliability and reproducibility of experimental results while reducing the time scientists require to perform bulk analysis of liver histology. This analytical process is readily adaptable by most laboratories, requires minimal optimization, and its principles and code can be optimized for use in other organs.Graphical overview
HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice
The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.
Intravital Imaging of Intestinal Intraepithelial Lymphocytes
Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol ofintravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.
Microbiology
Protein Structure Predictions, Atomic Model Building, and Validation Using a Cryo-EM Density Map from Hepatitis B Virus Spherical Subviral Particle
Hepatitis B virus (HBV) infection is a global public health concern. During chronic infection, the HBV small-surface antigen is expressed in large excess as non-infectious spherical subviral particles (SVPs), which possess strong immunogenicity. To date, attempts at understanding the structure of HBV spherical SVP have been restricted to 12–30 Å with contradictory conclusions regarding its architecture. We have used cryo-electron microscopy (cryo-EM) and 3D image reconstruction to solve the HBV spherical SVP to 6.3 Å. Here, we present an extended protocol on combining AlphaFold2 prediction with a moderate-resolution cryo-EM density map to build a reliable 3D model. This protocol utilizes multiple software packages that are routinely used in the cryo-EM community. The workflow includes 3D model prediction, model evaluation, rigid-body fitting, flexible fitting, real-space refinement, model validation, and model adjustment. Finally, the described protocol can also be applied to high-resolution cryo-EM datasets (2–4 Å).
Simplifying Barley Leaf Rust Research: An Easy and Reproducible Infection Protocol for Puccinia hordei on a Small Laboratory Scale
Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.Key features• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation• Plant age–independent protocol• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility• No specialized equipment needed• Includes simple spore harvesting method• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)• Protocol is also applicable for a detached leaf assayGraphical overview
Production and Purification of Cell Culture–generated Hepatitis B Virus by Transient Transfection and Density Gradient
An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line–derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture–generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.
Molecular Biology
In situ Quantification of Cytosine Modification Levels in Heterochromatic Domains of Cultured Mammalian Cells
Epigenetic modifications of DNA, and especially cytosine, play a crucial role in regulating basic cellular processes and thereby the overall cellular metabolism. Their levels change during organismic and cellular development, but especially also in pathogenic aberrations such as cancer. Levels of respective modifications are often addressed in bulk by specialized mass spectrometry techniques or by employing dedicated ChIP-seq protocols, with the latter giving information about the sequence context of the modification. However, to address modification levels on a single cell basis, high- or low-content microscopy techniques remain the preferred methodology. The protocol presented here describes a straightforward method to detect and quantify different DNA modifications in human cell lines, which can also be adapted to other cultured mammalian cell types. To this end, cells are immunostained against two different cytosine modifications in combination with DNA counterstaining. Image acquisition takes place on a confocal microscopy system. A semi-automated analysis pipeline helps to gather data in a fast and reliable fashion. The protocol is comparatively simple, fast, and cost effective. By employing methodologies that are often well established in most molecular biology laboratories, many researchers are able to apply the described protocol straight away in-house.
Neuroscience
An ex vivo Model of Paired Cultured Hippocampal Neurons for Bi-directionally Studying Synaptic Transmission and Plasticity
Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons.Key features• Preparation of ex vivo paired cultured hippocampal neurons.• Bi-directional electrophysiological recordings of synaptic transmission and plasticity.• Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel α2δ-2 subunit).Graphical overview
Plant Science
Inoculation of Maize with Sugarcane Mosaic Virus Constructs and Application for RNA Interference in Fall Armyworms
Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview
Relative Membrane Potential Measurements Using DISBAC2(3) Fluorescence in Arabidopsis thaliana Primary Roots
In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC2(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC2(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors.Key features• Non-invasive method to relatively quantify membrane potential in plant roots.• Method suitable for imaging seedlings root in agar or liquid medium.• Straightforward quantification.
Stem Cell
A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues
Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering.Key features• A new gastruloid culture system to model post-implantation mouse embryonic development in vitro• High-throughput formation of epiblast-like aggregates on hydrogel microwells• Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissuesGraphical overview
Systems Biology
Chromatin-RNA in situ Reverse Transcription Sequencing (CRIST-seq) Approach to Profile the Non-coding RNA Interaction Network
Non-coding RNAs (ncRNAs) are defined as RNAs that do not encode proteins, but many ncRNAs do have the ability to regulate gene expression. These ncRNAs play a critical role in the epigenetic regulation of various physiological and pathological processes through diverse biochemical mechanisms. However, the existing screening methods to identify regulatory ncRNAs only focus on whole-cell expression levels and do not capture every ncRNA that targets certain genes. We describe a new method, chromatin-RNA in situ reverse transcription sequencing (CRIST-seq), that can identify all the ncRNAs that are associated with the regulation of any given gene. In this article, we targeted the ncRNAs that are associated with pluripotent gene Sox2, allowing us to catalog the ncRNA regulation network of pluripotency maintenance. This methodology is universally applicable for the study of epigenetic regulation of any genes by making simple changes on the CRISPR-dCas9 gRNAs.Key features• This method provides a new technique for screening ncRNAs and establishing chromatin interaction networks.• The target gene for this method can be any gene of interest and any site in the entire genome.• This method can be further extended to detect RNAs, DNAs, and proteins that interact with target genes.Graphical overview