Name: stem cell-like xenopus embryonic explants to study early neural developmental features in vitro and in vivo
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Description: Watch this Scientific Journal Video about Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo at JoVE.com

The author thanks Hugo Juraver-Geslin, Marion Wassef and Anne H&#233;l&#232;ne Monsoro-Burq for their help and advice, and the Animal Facility of the Institut Curie. The author thanks Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the "Association pour la Recherche sur le Cancer" (ARC 4972 and ARC 5115; FRC DOC20120605233 and LABEX Memolife) and the Fondation Pierre Gilles de Gennes (FPGG0039).

Experiments comply with National and European regulation on the protection of animals used for scientific purposes and with internationally established principles of replacement, reduction and refinement.
1. Flat-mounting of Xenopus laevis Tadpoles Anterior Neural Tube After Whole-mount In Situ Hybridization
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	<li>Obtain X. laevis embryos according to standard procedures4 and age them until they reach neurula stage 26 and older (according to Nieuwkoo...

<ol>
	<li>Nieuwkoop, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IIB,+Pattern+formation+in+the+developing+central+nervous+system+(CNS)+of+the+amphibians+and+birds+(English).">IIB, Pattern formation in the developing central nervous system (CNS) of the amphibians and birds (English).</a> Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen. 94, 121-127 (1991).</li><li>Nieuwkoop, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+induction+process;+its+morphogenetic+aspects.">The neural induction process; its morphogenetic aspects.</a> Int J Dev Biol. 43, 615-623 (1999).</li><li>Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction.">Neural induction.</a> Curr Opin Genet Dev. 10, 357-362 (2000).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis : a laboratory manual. , (2000).</li><li>Hoppler, S., Vize, P. D., Hoppler, S., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus protocols : post-genomic approaches. , (2012).</li><li>Franklin Hughes, W., La Velle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+early+tectal+lesions+on+development+in+the+retinal+gonglion+cell+layer+of+chick+embryos.">The effects of early tectal lesions on development in the retinal gonglion cell layer of chick embryos.</a> J Comp Neurol. 163, 265-283 (1975).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beads+on+the+run:+beads+as+alternative+tools+for+chemotaxis+assays.">Beads on the run: beads as alternative tools for chemotaxis assays.</a> Methods Mol Biol. 769, 449-460 (2011).</li><li>Juraver-Geslin, H. 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M., Acosta, H., Gouignard, N., Climent, M., Arregi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+signals,+gradient+formation+and+regional+specificity+in+neural+induction.">Active signals, gradient formation and regional specificity in neural induction.</a> Exp Cell Res. 321, 25-31 (2014).</li><li>Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction:+old+problem,+new+findings,+yet+more+questions.">Neural induction: old problem, new findings, yet more questions.</a> Development. 132, 2007-2021 (2005).</li><li>Juraver-Geslin, H. A., Ausseil, J. J., Wassef, M., Durand, B. 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Neural development is orchestrated by a complex interplay between cellular developmental programs and signals from the surrounding tissues (Reviewed in3,31,32). Here we describe a set of protocols that can be used in X. laevis embryos to explore extrinsic and intrinsic factors involved in neural fate determination and neural morphogenesis in vitro and in vivo. These protocols can be used as such on X. tropicalis embryos, however X. tropicalis embryos are four times ...

The vertebrate nervous system emerges from the neural plate as a homogeneous layer of neuroepithelial cells. Understanding how developmental programs are induced, encoded, and established during regionalization of the neural plate is, at present, a major goal in developmental biology. Compared to other systems, the experimentally amenable Xenopus embryo is a model of choice for analyzing early steps of neural development1,2. It is easy to obtain large numbers of embryos, and external development gives access to the very first steps of neurulation3. Many tools are available to experimentally manipulate Xenopuslaevis (X. laevis) embryonic development. Micro-injection of mRNAs or morpholinos (MO), including inducible MOs, together with biochemical and pharmacological tools, allows controlled gain of function (GOF) and loss of function (LOF) and specific alteration of signaling pathways4,5. The blastocoel roof ectoderm, located around the animal pole of a blastula, or a very early gastrula embryo, and referred to as the &#39;Animal Cap&#39; (AC), is a source of pluripotent cells that can be programmed by manipulation of gene expression prior to explants preparation. In this manuscript are detailed protocols to use X. laevis AC explants to test in vitro and in vivo molecular mechanisms and cellular processes underlying neural development.
A technique is presented, allowing fine observation of gene expression patterns in a Xenopus tadpole neural tube, a preliminary step in the identification of fate determination cues. Whereas the observation of flat-mounted tissues is commonly used in the study of chick embryos6, it has not been properly described in Xenopus. Manipulation of gene expression by injecting synthetic mRNA or MO into the blastomeres of 2 or 4 cell stage embryos allows programming of AC explants4. For example inhibition of the Bone Morphogenetic Protein (BMP) pathway by expression of the anti-BMP factor Noggin, gives a neural identity to AC cells3. The protocol is detailed for performing local and time-controlled exposure of AC explants to extrinsic cues via direct contact with an anion exchange resin bead. Finally a technique is described for testing developmental features of neural progenitors in vivo by transplantation of mixed explants prepared from distinct programmed cells dissociated and re-associated.
The frog embryo is a powerful model to study early vertebrate neural development. Combining manipulation of gene expression to explant in vitro cultures provides important information in the study of neuroepithelium regionalization, proliferation, and morphogenesis7-12. The programming of AC explants permitted development of a functional heart ex vivo13,14. The use of explant grafting15 led to the identification of the minimal transcriptional switch inducing the neural crest differentiation program16. The zona limitans intrathalamica (ZLI) is a signaling center that secretes sonic hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. When continuously exposed to Shh, neuroepithelial cells coexpressing the three transcription factor genes - barH-like homeobox-2(barhl2),orthodenticle-2 (otx2) and iroquois-3 (irx3) - acquire two characteristics of the ZLI compartment: the competence to express shh, and the ability to segregate from anterior neural plate cells. As a model system, the induction of a ZLI fate into neuroepithelial cells will be presented8.
These protocols aim at providing simple, cheap, and efficient tools for developmental biologists and other researchers to explore the fundamental mechanisms of key neural cell behaviors. These protocols are very versatile and allow the investigation of a large range of extrinsic and intrinsic neural determination cues. It permits long term in vivo analysis of neural lineage commitment, inductive interactions and cell behaviors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>&#160;20909.290</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>anion exchange resin beads</td>
			<td>Biorad</td>
			<td>140- 1231</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>SIGMA</td>
			<td>A-7888</td>
			<td>For culture of animal cappH 7.6</td>
		</tr>
		<tr>
			<td>Gentamycine&#160;</td>
			<td>GIBCO</td>
			<td>15751-045&#160;</td>
			<td>antibiotic</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>&#160;for bead preparation</td>
		</tr>
	</tbody>
</table>

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.

Based on morphological considerations in different species, embryological manipulations, and the expression pattern of regulatory genes, a conceptual model holds that the neural plate is divided into transverse and longitudinal segments that define a developmental grid generating distinct histogenic fields. In the neural plate, the primordia of the forebrain, midbrain, hindbrain and spinal cord are all already established along the antero-posterior (AP) axis during gastrulation (reviewed ...

Watch this Scientific Journal Video about Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo at JoVE.com

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.

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, ...

The overall goal of this protocol is to use x laevis blastocoel roof or animal cap explants for testing the molecular mechanisms and cellular processes underlying neural development in vitro and in vivo. These methods can help answer key questions in the developmental and stem cell biology fields about fate acquisition, cell migration, and the cell autonomous and non-autonomous properties of neural cells. The presented techniques provide simple, cheap, versatile, and efficient tools for exploring the fundamental mechanism of key neural cells behaviors.These assays allow the testing of neural fate determinals for reprogramming induced pluripotent stem cells and the mature generation of neural derived cell ties furtherized screening. Generally, individuals new to this method will struggle as the techniques require manual dexterity. I first had the idea for these methods when I was thinking of analyzing cell segregation behavior.To flat-mount a xenopus laevis anterior neural tube use plastic transfer pipette to transfer a fixed, dehydrated and rehydrated embryo into a 60 millimeter petri dish filled to the top with PBT. Next, insert a pair of fine forceps between the eyes and the neural tube at the level of the optic stalk and detach the eyes. Then, carefulLy introduce the forceps below the ectoderm overlying the neural tube behind the head and carefully peel off the ectoderm.After performing the appropriate NC2 hybridization labeling transfer the embryo into a new 60 milliliter petri dish filled with PBT and use fine forceps to grossly isolate the anterior neural tube from the embryo. Then, carefully remove the loosely attached notochord from below the neural tube. If necessary, the otic vesicles and the remaining parts of the mesoderm.When all of the neural tube appendices have been severed, separate the anterior neural tube from the embryo. Then, transfer the isolated neural tube into a 1.5 milliliter conical tube filled with 50%glycerol and PBS for overnight treatment at four degrees Celsius. After a second overnight treatment in 90%glycerol, transfer the neural tube onto a microscope slide in a small amount of glycerol and use Tungsten needles to dissect the neural tubes along the dorsal and ventral midlines.Then use reinforcement rings covered with a glass cover slip to mount the two sides of the neural tube in 90%glycerol and PBS. To prepare bead for induction of animal cap explant use a P200 micropipette to split half an aliquot of BSA treated resin beads into a new 1.5 milliliter tube. Then, replace the sterile distilled water in the new tube with 200 microliters of medium containing the experimental molecule of interest for two hours at four degrees Celsius.At the end of the incubation, transfer blastula or very early gastrula embryos into PBS with calcium and magnesium supplemented with 0.2%BSA. Next, under a stereo microscope, pinch the vitelline membrane with the side of one pair of forceps and use a second pair of forceps to slowly peel the membrane from the vegetal side of the embryo taking care not to damage the animal side. Then, using fine forceps, cut out a small square from the animal pole of each embryo.Using a BSA-coated pipette tip, transfer the caps into individual wells of a Terasaki multiwell plate in 0.5X modified Barth's saline, placing the pigmented animal side in contact with the round bottom of the well. Using a P20 micropipette, select the darkest pink beads from the treatment tube and place one bead onto each cap, using forceps to center the beads on the caps. When the last bead has been placed, set the plate on top of water-soaked papers and cover the plate with a plastic container before incubation.To prepare dissociated and reaggregated animal cap explants, isolate 15 to 30 animal caps as just demonstrated. Using fine forceps to cut out a small square of tissue from the animal poles. Transfer the animal caps into an agarose coated well filled with calcium-free Holtfreter saline with their pigmented sides facing up.After a few minutes, disaggregation should be observed. Use fine forceps to separate the pigmented layers from the rest of the tissue. And then, use a P20 micropipette to discard the pigment.When most of the pigmented layers have been removed and the cells are completely dissociated, swirl the plate to center the cells and use a P1000 micropipette to remove as much of the medium as possible, taking care not to touch the cells. Then, add one milliliter of Holtfreter's saline with calcium to the well. Transfer the dissociated animal caps into a 1.5 milliliter tube.After spinning down the cells, use a P1000 micropipette to carefully remove the supernatant and re-suspend the pellet in 20 microliters of Holtfreter's saline with calcium. Allow the cells to reaggregate for three to six hours at room temperature. Then slowly add one milliliter of 3/4 NAM to carefully detach the explants from the bottom of the tube and use a plastic transfer pipette to distribute the explants into individual uncoated plates.To explant animal cap explants onto xenopus laevis neural plates, first coat 60 millimeter petri dishes with 3%agarose and 3/4 NAM into which small holes have been made. Next, transfer early neurula embryos into PBS with calcium and magnesium supplemented with a 0.2%BSA for removal of the vitelline membrane. Then fill the agarose-coated dissection dish with fresh 3/4 NAM and transfer the embryos into the dissection dish dorsal side up.Now, transfer an animal cap reaggregated explant to the dissection plate allowing the explant to slowly sink down into the dish by gravity once the pipette tip has breached the NAM surface. Then under a stereo microscope, use rounded forceps to hold the embryo in place while using a Tungsten needle to make an incision on the neural plate for the explant. Cut out a small piece of the reaggregated animal cap explant using rounded forceps and an eyebrow knife.Use the tissue fragment to size a piece of explant for the graft. This figure shows X laevis and X tropicalis anterior neural tubes flat-mounted as just demonstrated at stages 30, 32, and 35 after whole mount double NC2 hybridization. As observed in these images, the expression of xenopus sonic hedgehog is detected in cells in contact with beads soaked in conditioned medium supplemented with the end terminus of the sonic hedgehog gene, but not in cells in contact with beads treated with control medium.After dissociation and reaggregation as just demonstrated, these explants with anteriorized animal cap cells were mixed with anteriorized animal cap cells expressing Otx2. As observed, these Otx2 expressing cells did not segregate from the non-Otx2 expressing cells. Both types of cells intermingled freely throughout the explant.In contrast, in explants where Otx2 expressing cells were mixed with BAR HL-2, Otx2 and IRX3 expressing cells the multigene expressing cells formed cohesive patches that did not spread uniformly within the reaggregated explant. When grafted into the anterior neural plate of x laevis embryos as just demonstrated, mixed explants composed of cells coexpressing BAR HL-2, Otx2, and IRX3 mixed with N-terminus sonic hedgehog expressing cells exhibited indogenous sonic hedgehog expression and segregated from the anterior neural epithelial cells. Whereas, when mixed explants composed of Otx2 and N-terminus sonic hedgehog expressing cells were grafted into the x laevis neural plate, the grated cells neither expressed sonic hedgehog nor segregated from their neighbors.Once mastered, grafting the animal cap explant onto embryonic neural plates can be performed in two to three hours depending on the number of embryos grafted. It is a very robust and very rewarding technique. When attempting this procedure, it's important to be extremely cautious about bacterial and fungal contamination.Following the grafting procedures, other methods like whole mount in situ hybridization can be performed to follow the fates and the behaviors of the grafted cells. The developmental programs underlying neural tube organogenosis are largely conserved especially in vertebrates. Thus, information acquired in amphibians has furthered our understanding of the cellular and molecular processes underlying vertebrate development.

stem-cell-like-xenopus-embryonic-explants-to-study-early-neural

Research

JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

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 ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

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 ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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 ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...