Name: light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development
Uploaded: 2017-06-15T15:00:00
Description: Watch this Scientific Journal Video about Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development at JoVE.com

This work was supported by the University of Illinois at Urbana-Champaign (UIUC) and the National Institutes of Health (NIGMS R01GM111816).

Animal research was conducted in accordance with guidelines set by the Illinois Institutional Animal Care and Use Committee (IACUC) and the University of Illinois Department of Animal Resources (DAR).
1. Optogenetic Induction of Protein Localization in BHK21 Mammalian Cell Culture
NOTE: Steps 1.1-1.3 provide a method to assemble a cell culture chamber for imaging with high-magnification objectives (e.g., 63X or 100X), which typically have short working distan...

When building the light box, the power of individual LEDs should be measured. Based on previous experience, the power output can vary between individual LEDs due to manufacturing variance. Select a set of LEDs that have a power output within 10% of each other. The number of LEDs, the current-limiting resistor, and the power input can be modified for different types of cell culture containers (e.g., a 6-well or 24-well plate). A 24 h of light illumination at a power of 0.2 mW/cm2 does not induce detect...

Growth factors are involved in a wide spectrum of cell functions, including proliferation, differentiation, migration, and apoptosis, and play pivotal roles in many biological events, including embryonic development, aging, and regulation of mental status1,2,3,4,5. Many growth factors signal through complex intracellular signaling cascades. These signaling events are often operated by reversible protein phosphorylation in a precisely regulated fashion6,7. Thus, an understanding of the signaling outcomes of protein kinases, which are responsible for protein phosphorylation, is fundamentally important.
Different growth factors act through a rather common intracellular signaling network, even though they stimulate distinct cellular responses8,9. Common intracellular mediators of receptor tyrosine kinases include Ras, Raf, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), phosphoinositide 3-kinase (PI3K), Akt, and phospholipase C gamma (PLC&#947;)10,11. Accumulating evidence suggests that signaling diversity and specificity depend upon the spatial and temporal regulation of signaling activity12. For instance, in rat pheochromocytoma cells (PC12), epidermal growth factor (EGF) stimulation, which results in cell proliferation, transiently activates the ERK pathway9. On the other hand, stimulation with nerve growth factor (NGF), which leads to cell differentiation, activates the ERK pathway in a sustained manner9,13. In cultured rat hippocampal neurons, transient signaling by brain-derived neurotrophic factor (BDNF) promotes primary neurite outgrowth, while sustained signaling leads to increased neurite branching14. During early embryonic development, phosphorylated ERK activity is temporally dynamic and is widespread across the embryo6. A recent genetic screen during early Xenopus embryogenesis showed that ERK and Akt signaling cascades, two downstream primary growth factor pathways, display stage-specific activation profiles7. Thus, an understanding of kinase signaling outcomes calls for tools that can probe the spatial and temporal features of kinase activity with sufficient resolution.
Conventional experimental approaches to probe the dynamic nature of signal transduction during development lack the desirable spatial and temporal resolution. For instance, pharmacological approaches utilize small chemical or biological molecules to stimulate or suppress signal transduction in cells and tissues. The diffusive nature of these small molecules makes it challenging to restrict their action to a specific region of interest15. Genetic approaches (e.g., transgenesis, the Cre-Lox system, or mutagenesis) often lead to the irreversible activation or repression of the target gene expression or protein activity16,17,18. The Tet-On/Tet-Off system19 offers improved temporal control of gene transcription but lacks strict spatial control because it relies on the diffusion of tetracycline. Recent developments in chemically induced protein dimerization20 or photo-uncaging21,22,23,24 have greatly enhanced the temporal control of signaling networks. The spatial control, however, remains challenging due to the diffusive nature of the caged chemicals.
Recent emerging optogenetic approaches, which harness the power of light to control protein-protein interactions, allow for the modulation of signaling pathways with high spatiotemporal precision as well as reversibility. Shortly after its initial success in controlling neuronal firing25,26,27, optogenetics has been extended to control other cellular processes, such as gene transcription, translation, cell migration, differentiation, and apoptosis28,29,30,31,32,33,34. A strategy using the photoactivatable protein pair Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN) was recently developed to control Raf1 kinase activity in mammalian cells and Xenopus embryos35. CRY2 binds to CIBN upon blue-light stimulation, and the CRY2/CIBN protein complex dissociates spontaneously in the dark34. Blue light excites the CRY2 cofactor, flavin adenine dinucleotide (FAD), which leads to a conformational change in CRY2 and its subsequent binding to CIBN. Constitutively active (W374A) and flavin-deficient (D387A) mutants of CRY2 can be produced through mutations in the FAD-binding pocket: the CRY2W374A mutant binds to CIBN independent of light, whereas the CRY2D387A mutant does not bind to CIBN under blue-light stimulation36,37. The optogenetic system described in this protocol uses wild-type CRY2 and CIBN to induce protein translocation-mediated Raf1 activation in live cells. It is known that the membrane recruitment of Raf1 enhances its activity38. In this system, a tandem CIBN module is anchored to the plasma membrane and CRY2-mCherry is fused to the N-terminal of Raf135. In the absence of blue light, CRY2-mCherry-Raf1 stays in the cytoplasm, and Raf1 is inactive. Blue-light stimulation induces CRY2-CIBN binding and recruits Raf1 to the plasma membrane, where Raf1 is activated. Raf activation stimulates a Raf/MEK/ERK signaling cascade. Both CRY2- and CIBN- fusion proteins are encoded in a bicistronic genetic system. This strategy can be generalized to control other kinases, such as Akt, whose activation state can also be turned on by protein translocation in cells39. This work presents detailed protocols for implementing this optogenetic strategy in mammalian cell cultures and multicellular organisms.

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light-mediated reversible modulation of the mitogen-activated protein kinase pathway during cell differentiation and xenopus embryonic development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Ratiometric expression of photoactivatable protein pairs: Figure 1A shows the design of a bicistronic optogenetic construct, CRY2-mCherry-Raf1-P2A-CIBN-CIBN-GFP-CaaX (referred to as CRY2-2A-2CIBN), based on the porcine teschovirum-1 2A (P2A) peptide, which shows the highest ribosome-skipping efficiency among mammalian cell lines42. In previous work, it has been determined that the optimal ratio for CIBN-GFP-CaaX:CRY2-mCherry-Raf1 is 2:...

Watch this Scientific Journal Video about Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development at JoVE.com

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

The overall goal of this experiment is to use light to control the mitogen-activated protein kinase signaling pathway in intact cells and in Xenopus embryos. This method can help answer key questions in cellular and developmental biology, such as how temporal modulations of kinase activity regulates self-determination during cell differentiation, and embryonic development. The main advantage of this technique is that the mitogen-activated protein kinase activity can be controlled reversibly in intact cells and in multicellular organisms, such as developing embryos.This method can provide insight into signaling kinetics during Xenopus embryonic developments. It can also be applied to other model systems such as socotra, zebrafish, or mouse. Today, a graduate student from my laboratory, Vishnu Krishnamurthy, will demonstrate the procedure.And Aurora Turgeon, a post-star in my laboratory, will demonstrate all procedures involved in Xenopus embryos. First, assemble the cell culture device by placing one autoclaved sterile PDMS chamber on a sterile PLL-coated cover slip in a 60 mm petri dish. Next, use 0.5 mL of 0.25%trypsin to detach pHK21 cells from one well of a 6-well tissue culture plate.After counting the cell density with the hemocytometer, seed the chamber with 20, 000 cells in 200 microliters of cell culture medium. 24 hours after cell plating, transfect the cells with 50 to 100 nanograms of CRY2-mCherry-Raf1 2A2CIBMG of PCaA X plasmid as per the manufacturer's protocol. Three hours after transfection, change the medium to 200 microliters of fresh culture medium and allow the cells to recover overnight By incubating the culture in a 37 degree Celsius and five percent carbon dioxide incubator.The next day, begin optogenetic induction and imaging by first replacing the cell culture medium with 200 microliters of carbon dioxide independent medium. Then set up the data acquisition protocol. Select 488 nonometer excitation FITC channel for optogenetic stimulation, and the 561 nanometer excitation CRITC channel to track the cellular localization of mCherry labeled protein.Measure the power of the blue light by placing a power meter close to the objective window. A total power of two microwatts is sufficient to induce CIBN CRY2-PHR association. Set up a time stamp acquisition with the five second interval.And the total acquisition time of two minutes. Apply appropriate index-matching material on the objective window. Place the cell chamber on the microscope stage and use the bright field mode to focus on the cells on the coverslip surface using the eye pieces.Next, move the microscope stage to locate a transfected cell under green light. Record a series of time stamped images in both the FITC and CITC channels. The basic sample image shows CIBNGPCAAX localized on the plasma membrane.This is a snapshot of CRY2 mCherry raf1 before blue light simulation, whereas this images shows a snapshot of CRY2 mCherry raf one after ten pulses of blue light stimulation. Make the LED array by inserting tonal blue LEDs into two breadboards and connecting with current limiting resistors. Place the breadboards into an aluminum box.Insert two metal wires to connect the boards to the power supply. Make sure that the length of the wire is sufficient when the light box is placed inside a carbon dioxide incubator. Then place a transparent light diffuser to act as the cover of the light box.Lastly, calibrate the power output of each LED at a range of voltage inputs. Use a power of zero point two milliwatts per square centimeter for the 24 hour PC12 cell differentiation assay. Place the LED array in a 37 degree Celsius incubator supplemented with five percent carbon dioxide.Connect it to the power supply using the metal wires and set the power of the LED to zero point two milliwatts per square centimeter. Place the 12 well plate containing transfected PC12 cells on the window of the LED array. Align the plate so that every well is fully illuminated.Apply continuous light illumination for 24 hours. To image the differentiated cells, set up single snapshot data acquisition. Use 200 milliseconds for both the GFP and TexasRed channels.Capture images of the transfected cells in both the GFP and TexasRed channels, and save the files for data analysis. Begin this procedure by fabricating needles for RNA microinjection by pulling glass capillaries with a capillary puller. Remove the jelly coat from the embryos by treating them with three percent cysteine diluted in 0.2 XMMR for 15 minutes.Transfer the embryos to three percent polysucrose And zero point five XMMR solution for microinjection. Accurately inject 500 picograms to one nanogram of CRY2-mCherry-Raf1-2A-2CIBN-GFP-CaaX RNA into each embryo. Then culture the microinjected embryos in the microinjection solution at room temperature.When the embryos reach the med gastrula stage, transfer them to 0.2x MMR solution, and continue to culture until the desired stage of development. Then place the 12-well plate on the home built LED array for blue light treatment. Place a mirror on top of the 12-well plate to ensure full blue light illumination of the embryos.Harvest the embryos for histological, western blot, or gene expression analysis after exposure to blue light for the desired time. This image shows multichannel snapshots of PC12 cells tranfected wth CRY2-2A-2CIBN after 24 hours of blue light stimulation at zero point two milliwatts per square centimeter. Both the GFP and mCherry reporters are seen.Circles mark differentiated cells and squares mark undifferentiated cells. Here the cells were treated the same way as those in the prior image, except no blue light stimulation was used. No differentiation is seen.These cells show representative results after transfection with raf1 GFPCaaX, a constitutively active raf1. The circles and rectangles mark differentiated and undifferentiated cells respectively. This histogram shows differentiation ratios of PC12 cells transfected with CA-Raf1, Co-transfected with CRY2 mCherry raf1 and CIBN GFP Caax and singly transfected with CRY2A 2CIBN.Only cells exposed to blue light showed significant differentiation. This image shows the morphology of normal Xenopus embryos without MRNA injection, and without light treatments. This image shows embryos injected with CRY2 2A2CIBN MRNA and subjected to blue light stimulation.Activation of raf1 by treating CRY2 2A2CIBN injected embryos with blue light induces ectopic tail-like structures in the head region. Following this procedure, one can answer additional questions, such as how stage specific activation of the mitogen activated protein kinase pathway regulates gene expression. This optogenetic method can be generalized to control other signaling pathways with similar activation mechanisms.

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Unit 1

Signaling Networks of Kinase Receptors

SCIENTISTS IN ACTION

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...