CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1. Electroporate the Construct of Interest in the Neural Tubes of HH12-13 Chicken Embryos In ovo
Use a fluorescent reporter, preferably in a bicistronic transcript or fused to the protein of interest with an intercalating viral 2a peptide11, to enable quick identification of individuals with satisfactory levels of transfection. NOTE: Typical incubation time for this stage is around 48 hr at 37.8 &#176;C.
<ol>
	<li>Open a small window in the eggs after removing 3 ml of albumin with a syringe coupled to a 18 G x 1 1/2 needle. To enhance embryo visualization, inject a small amount of Indian ink diluted 1:50 in sterile Ringer&#8217;s solution underneath the embryo1-4.</li>
	<li>After covering the embryo with sterile Ringer&#8217;s solution, inject a 10 mM Tris-HCl, pH 8.2 and 0.1% Food Dye (F, D &#38; C) solution containing 2.5 &#181;g/&#181;l DNA through the posterior end of the neural tube until it fills up to the hindbrain region. To electroporate, position platinum electrodes (0.5 mm in diameter) separated by 4 mm1 along the flank region and deliver 5 pulses of 50 msec and 20 V with an interval of 100 msec between them4.</li>
	<li>After electroporation, incubate manipulated embryos for 48 hr more until they reach stage HH23 and identify individuals presenting high level of transfection at the flank region under a fluorescent stereomicroscope. 
	NOTE: The duration of incubation after electroporation depends on the aim of the experiment. The electroporated plasmids can generate significant expression levels after 2 hr2. Thus, if the interest lies in early onset genes the post-electroporation incubation period can be reduced.</li>
</ol>
2. Harvest Successfully Transfected Embryos and Dissect the Electroporated Spinal Cord Halves
NOTE: This procedure lasts from 2-3 hr for each 10 embryos and yields 1-2 &#181;g total RNA per embryo. The dissection of neural tubes requires fine movements and some practice sessions may be necessary before applying it to experimental samples.
<ol>
	<li>Decontaminate metallic dissection tools with RNase decontamination solution or by baking at 150 &#176;C for 4 hr12 and prepare DEPC-treated PBS. Use only new certified RNase-free plasticware. 
	NOTE: After harvesting, it is important to follow standard care to prevent RNase contamination during dissection.</li>
	<li>Harvest the embryos using a pair of fine dissecting scissors and a sieved collection spoon. Keep the embryos in cold DEPC-treated PBS in a 100 mm Petri dish.</li>
	<li>With a pair of fine tweezers, cut out the whole flank region (between the posterior limit of the anterior limb bud and the anterior limit of the posterior limb bud) and transfer this section to a new dish containing cold DEPC-treated PBS.</li>
	<li>Use two fine glass needles to perforate at three different points on each dorsolateral side of the embryo between the neural tube and the paraxial mesoderm.</li>
	<li>Introduce the two glass needles in the central perforation and slowly move one of them away along the neural tube anterior-posterior axis. Repeat this for the perforations that were not reached by the first movements. Remove the detached paraxial mesoderm with a pair of fine tweezers.</li>
	<li>Turn the embryo sideways, insert the tips of the tweezers between the neural tube and the notochord and slide them in opposite directions along the anterior-posterior axis to separate the two structures. 
	NOTE: This should also remove most of the mesoderm still attached to the neural tube.</li>
	<li>If necessary, remove any larger pieces of mesodermic tissue that remain attached by pulling with a pair of tweezers while lightly holding the neural tube with another pair of tweezers. When finished, gently transfer the neural tube to another dish containing cold DEPC-treated PBS using a RNase-free glass Pasteur pipette coupled to a manual pipette pump and keep on ice until all the tubes are dissected.</li>
	<li>Divide the neural tubes into two halves. For this, insert the sharp tip of a closed pair of tweezers in the lumen and open the roof plate by moving the tweezers out through it. After this, separate the halves by cutting the floor plate with the tweezers tips.</li>
	<li>Sort electroporated from non-electroporated halves under a fluorescent dissecting scope and transfer to a 1.5 ml tube filled with ice-cold DEPC-treated PBS. If necessary, flick the microtubes gently to release any tissue attached to the plastic walls and to promote sedimentation of the tubes.</li>
	<li>Spin at 100 x g for 3 sec and remove very slowly as much PBS as possible with a 1 ml micropipette. Do this while looking at a light source that shines through the microtube to monitor if any embryonic material is sucked to the pipette tip; if this happens, slowly discharge and repeat the centrifugation.</li>
	<li>Add 70 &#181;l of lysis solution for RNA extraction per neural tube half and flick the tube to mix contents. Incubate at room temperature for 5 min and vortex vigorously. Repeat the 5 min incubation and vortexing two more times. Store at -80 &#176;C.</li>
	<li>Collect at least eight neural tube halves per sample to ensure an adequate yield for the next step. Pool smaller samples if several electroporation/dissection sessions are required to amass sufficient number of neural tubes. Store sufficient samples for duplicates or triplicates for each condition.</li>
</ol>
3. Purify RNA and Prepare Libraries for Illumina Sequencing
<ol>
	<li>Remove samples from -80 &#176;C storage and place on ice for 15-30 min. Resume thawing at room temperature until complete.</li>
	<li>Vortex well and leave at room temperature for 5 min. Vortex again and centrifuge at 15,000 x g for 1 min to pellet insoluble material.</li>
	<li>Transfer the supernatant to a new tube and proceed to RNA extraction according to the total RNA isolation kit manual instructions. Elute in the minimum recommended volume to increase concentration. 
	NOTE: DNase treatment after clean-up is recommended in order to avoid potential contamination of genomic DNA sequences in RNA-Seq datasets.</li>
	<li>Flick the tube to mix contents and remove 5 &#181;l for quality control analysis. Store the remaining sample at -80 &#176;C until library preparation; these samples should be thawed only once to avoid RNA degradation.</li>
	<li>Run purified RNA in a Bioanalyzer instrument using the RNA 6000 Nano kit; RIN value must be at least above 7 to proceed.</li>
	<li>Generate libraries using an Illumina compatible RNA-Seq library preparation kit and multiplex up to 4 libraries in one lane of a HiSeq instrument set to generate 100 bp single reads. If possible, include all samples in the same run to minimize potential technical artifacts. 
	NOTE: This should yield up to 50 million reads per sample, which is more than enough to obtain coverage for most genes13.</li>
</ol>
4. Compare RNA-Seq Datasets in the Galaxy Public Server10.
NOTE: Server storage space is limited and experiments comparing many samples may require deletion of intermediate files generated in the filtering steps.
<ol>
	<li>Create a user account in the Galaxy public server (<a href="https://usegalaxy.org/">https://usegalaxy.org</a>) and FTP upload the Illumina datasets in fastq format.</li>
	<li>Prepare uploaded files with the FastqGroomer14 tool under the NGS:QC and manipulation menu; if the quality scores are already encoded in Phred+33 (Sanger), skip this step by simply changing the file type to fastqsanger under the Attributes for each dataset.</li>
	<li>Analyze the general quality of each dataset using the FastQC tool under the NGS:QC and manipulation menu. 
	NOTE: Special attention should be given to the resulting graphics for per base sequence quality and per base sequence content, as these will provide estimates for the parameters used for trimming the ends of the reads.</li>
	<li>Trim reads to discard 3&#8217; ends with low quality data using the tool FASTQ Quality Trimmer14 under the NGS:QC and manipulation menu. Set the tool to process only 3&#8217; ends for quality score below 20.</li>
	<li>Remove adapter sequences from reads using the Clip tool (<a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a>) under the NGS:QC and manipulation menu. Set the tool to discard reads with less than 45 bp left, input the sequence of the adapter used and choose the output to include both clipped and non-clipped sequences. Additionally, opt not to discard sequences with unknown bases since most low quality bases were already removed in the last step.</li>
	<li>Using the tool Trim sequences (<a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a>) under the NGS:QC and manipulation menu, trim the first bases of the reads to remove 5&#8217; sequence bias detected by FastQC (typically 10). 
	NOTE: At this point, it is a good idea to run FastQC again and compare with the previous analysis to determine if the dataset quality was improved.</li>
	<li>Download and unpack the latest chicken genome assembly for the UCSC genome browser from Illumina iGenomes (<a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a>) and upload the whole genome fasta format file and the genes annotation gtf format file to the Galaxy history using the tool Upload File under the Get Data menu.</li>
	<li>Map reads for each sample to the chicken genome using the aligner TopHat215 under the NGS: RNA Analysis menu. Choose to use a genome from History (select the fasta file uploaded in step 4.7).</li>
	<li>Build a predicted transcriptome for each sample using Cufflinks16 under the NGS: RNA Analysis menu. Choose to use reference annotation as a guide (select the gtf file uploaded in step 4.7) and turn on the options Perform Bias Correction (from History, fasta file from step 4.7) and Use multi-read correct.</li>
	<li>Merge predicted transcriptomes for all samples using Cuffmerge16 under the NGS: RNA Analysis menu. Choose to use reference annotation (gtf file from step 4.7) and sequence data (from History, fasta file from step 4.7).</li>
	<li>Compare BAM files generated by TopHat2 for each sample using Cuffdiff16 under the NGS: RNA Analysis menu. In the field Transcripts select the merged transcriptome generated by Cuffmerge and turn on the options Perform Bias Correction (from History, fasta file from step 4.7) and Use multi-read correct.</li>
	<li>Sort differential expression testing tables numerically ascending in column 13 (q value) using the tool Sort under the Filter and Sort menu. 
	NOTE: Genes/transcripts showing significant differential expression between experimental conditions will present the lowest q values.</li>
	<li>To check results visually with coverage graphs, first convert BAM files to bedGraph format using the tool Create a BedGraph of genome coverage17 under the BEDTools menu. Disable reporting of regions without coverage, choose to treat spliced entries as distinct intervals and scale the coverage by a factor that normalizes sequencing depth for all the datasets being compared.</li>
	<li>Convert the resulting bedGraph file to bigWig format using the tool Wig/BedGraph-to-bigWig (<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a>) under the Convert Formats menu. Upload the bigwig files as custom tracks in the UCSC genome browser (<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a>) for the galGal4 assembly and search a gene of interest to visualize the coverage in the datasets.</li>
</ol>

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The authors have nothing to disclose.

Here we provide guidelines for analyzing effects after electroporation of the chicken spinal cord. Although electroporation of DNA vectors is more frequently used to overexpress a gene of interest, one can also use constructs encoding dominant negatives, quimeric proteins or precursors for siRNAs to generate gene function knock-down conditions19-21. In fact, comparison of transcriptome profiles resulting from both gain- and loss-of-function analysis may point out genes differentially expressed in opposite ways...

Genetic studies on live organisms frequently use the chicken embryo as a model because in ovo electroporation represents a fast and inexpensive way to partially transfect embryonic structures in vivo with DNA constructs1-5. Bicistronic expression vectors that encode one transcript containing a fluorescent reporter together with the gene of interest, such as pCIG6 or pMES7, allow quick verification of transfection quality in the desired region under a stereomicroscope before processing embryos for downstream analysis.
With recent advances in DNA sequencing, it is now possible to obtain digital whole transcriptome expression profiles from RNA samples using RNA-Seq8,9. Therefore, instead of time-consuming methods that enable analysis of only a few gene products in parallel, such as in situ hybridization, immunohistochemistry and qPCR, preparation of cDNA libraries for high-throughput sequencing can provide information of the whole transcriptome. Observation of the experimental effects on the expression levels of every single gene may in turn provide powerful insights on the pathways altered by the construct used. This is particularly easy if a genomic assembly for the organism used is available, thus the chick embryo again is a suitable model.
If the user has access to a reliable genome sequencing center, the main issue is obtaining high quality RNA samples. This work demonstrates a method for obtaining RNA samples from transfected neural tubes suitable for RNA-Seq, as well as the downstream steps for in silico analysis of the resulting datasets. After electroporation at HH12-13 followed by 48 hr incubation, transfected trunk spinal cord halves were dissected in ribonuclease-free conditions, immediately lysed and further protected from degradation by ultra-low freezing until RNA purification and library preparation.
The Galaxy public server10 provides access to free resources for analyzing high-throughput sequencing data. The amount of space offered for registered users is enough for small experiments, thus eliminating the need for local computational infrastructure. RNA-Seq data analysis includes filtering, alignment and quantitation/comparison of gene expression. Although there are general guidelines for RNA-Seq data analysis, filtering parameters will largely depend on the quality of sequencing and should be determined after quality control analysis of the raw data.
This protocol describes the steps to obtain and compare RNA-Seq profiles from chicken embryonic spinal cords transfected in vivo. As a representative result, comparison of datasets obtained from two independent control samples transfected with an empty vector showed that the method is reproducible and should allow identification of differentially expressed transcripts in experimental samples. Therefore, application of this method has the potential to greatly increase the number of discoveries after one single experiment and thus provide a large set of data for subsequent investigation.

<table><tbody><tr><td>Indian ink</td><td>Any supplier</td><td></td><td></td></tr><tr><td>18G x 1 1/2 needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>3 ml syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Tris</td><td>MP Biomedicals</td><td>819620</td><td></td></tr><tr><td>F D &amp;amp; C Blue 1</td><td>Spectra Colors Corporation</td><td>5.FC.0010P0</td><td>Food Dye used to visualize DNA injection</td></tr><tr><td>Ringer&#039;s solution (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 7.2 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.37 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.225 g CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>10043-52-4</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.217 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.02 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Filter and autoclave</td><td></td><td></td><td></td></tr><tr><td>PBS (Phosphate Buffered Solution) (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 8 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1.15 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.2</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L and filter</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1 ml DEPC (Diethylpyrocarbonate)</td><td>Sigma-Aldrich</td><td>D-5758</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Shake vigorously and let stand overnight at room temperature</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Autoclave</td><td></td><td></td><td></td></tr><tr><td>Sieved spoon</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Sterile 60 x 15 mm polystirene Petri dishes</td><td>Corning Life Sciences</td><td>351007</td><td></td></tr><tr><td>RNaseZap RNase decontamination solution</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td>Fine point surgical scissors</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Straight fine point tweezers</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td><td>Kimble Chase</td><td>40A502</td><td></td></tr><tr><td>9&#039;&#039; glass Pasteur pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Manual pipette pump</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Clear 1.5 ml microcentrifuge polypropilene tubes</td><td>Corning Life Sciences</td><td>MCT-150-C</td><td></td></tr><tr><td>Microcentrifuge</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNAlater solution for RNA stabilization</td><td>Life Technologies</td><td>AM7020</td><td></td></tr><tr><td>RNAqueous total RNA isolation kit</td><td>Life Technologies</td><td>AM1912</td><td></td></tr><tr><td>Molecular Biology Grade Ethanol</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>2100 Electrophoresis Bioanalyzer Instrument</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>Truseq RNA Sample Preparation Kit v2</td><td>Illumina</td><td>RS-122-2001/RS-122-2002</td><td></td></tr></tbody></table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

To validate the method described, two control samples were generated from embryos electroporated with the empty vector pMES7 and one sample from embryos transfected with the same vector containing the insert construct SCRT2-ZNF, which encodes the zinc-finger domains of chicken SCRT218. Samples yielding 11-22 &#181;g total RNA each were obtained from pools of 8 to 12 embryos. All three samples scored an optimal RIN value of 10 in a quality check performed with the Bioanalyzer instrument (Figu...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.5K Views.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Video: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Genetics

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Developmental Biology

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Biology

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Summary

Explore More Videos

Chapters in this video

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo