Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

The animal part of this study was conducted at the Metabolic Research Unit of the University of Alberta, Edmonton, Canada, with all animal experimental procedures approved (Protocol # AUP00000131) by the University of Alberta Animal Care and Use Committee, and animals cared for according to the Canadian Council of Animal Care guidelines (1993).
1. Embryo Production, Isolation of Total RNA and DNase Treatment
<ol>
	<li>For animal experimental protocols and embryo collection refer in our previous publications17, 18.</li>
	<li>Pool and snap freeze similar stage embryos from each cow in liquid nitrogen and then keep at -80 &#176;C until RNA extraction.</li>
	<li>Extract total RNA via a column-based RNA extraction kit which enables to extract RNA from pico-scale samples (kit information is available in Material List). 
	NOTE: Recommended kit contains: Conditioning Buffer, Extraction Buffer, 70% Ethanol, Wash Buffer 1, Wash Buffer 2, Elution Buffer, RNA purification columns with collection tubes and microcentrifuge tubes.</li>
	<li>Add 10 &#181;L Extraction Buffer to tube containing embryos and incubate for 30 min at 42 &#176;C. Centrifuge tube at 800 x g for 2 min to collect cell extract into the microcentrifuge tube.</li>
	<li>Pipette 250 &#181;L Conditioning Buffer onto the purification column filter membrane. Incubate the RNA purification column with Conditioning Buffer for 5 min at room temperature. Centrifuge the purification column in the collection tube at 16,000 x g for 1 min.</li>
	<li>Pipette 10 &#181;L of 70% Ethanol into the mixture of RNA Extraction Buffer and embryos. Mix well by pipetting up and down. Pipette mixture into the preconditioned purification column.</li>
	<li>To bind RNA, centrifuge the purification column for 2 min at 100 x g and immediately follow by a centrifugation at 16,000 x g for 30 s to remove flow through.</li>
	<li>Pipette 100 &#181;L of Wash Buffer 1 into the purification column and centrifuge for 1 min at 8,000 x g. 
	NOTE: DNase treatment is recommended if performing reverse transcription or amplification after RNA isolation.</li>
	<li>Digest genomic DNA right after step 1.8. 
	NOTE: Recommended DNase kit is available in Material List. Recommended kit contains: DNase I stock solution and Buffer.</li>
	<li>Prepare DNase incubation mixture by mixing 5 &#181;L DNase I stock solution with 35 &#181;L Buffer. Mix by gently inverting.</li>
	<li>Pipette the 40 &#181;L DNase incubation mixture directly into the purification column membrane. Incubate at room temperature for 15 min.</li>
	<li>Pipette 40 &#181;L Wash Buffer 1 into the purification column membrane and then centrifuge at 8,000 x g for 15 s.</li>
	<li>Pipette 100 &#181;L Wash Buffer 2 into the purification column and centrifuge for 1 min at 8,000 x g.</li>
	<li>Pipette another 100 &#181;L Wash Buffer 2 into the purification column and centrifuge for 2 minutes at 16,000 x g.</li>
	<li>Transfer the purification column to a new 0.5 mL microcentrifuge tube. Pipette 11 &#181;L of Elution Buffer directly onto the membrane of the purification column. To ensure maximum absorption of Elution Buffer into the membrane, gently touch the tip of the pipette to the surface of the membrane while dispensing the elution buffer.</li>
	<li>Incubate the column for 1 min at room temperature. Centrifuge the column for 1 min at 1,000 x g to distribute Elution Buffer in the column, and then spin for 1 min at 16,000 x g to elute RNA.</li>
	<li>Use bioanalyzer instrument according to the manufacturer&#39;s instructions to evaluate the RNA quality and quantity19.</li>
	<li>Store the RNA sample at -80 &#176;C until use.</li>
</ol>
2. RNA Amplification and Labeling for Microarray Analysis
NOTE: For the first time user working with RNA amplification and labeling procedures, use five ng of spike-in RNA along with the actual aRNA samples to monitor the quality control measurement of the RNA amplification and hybridization. The spike-in RNA mix contains ten in vitro synthesized, polyadenylated transcripts in predetermined ratios. When the procedure performed properly, the labeled transcripts specifically hybridize only to complementary control probes in arrays and the data can be scanned to track quality control assurance with minimal self- or cross-hybridization. More details description regarding the spiked-in control intensities and normalization procedure after scanning the array and data analysis is refer to previous publications20, 21. The following procedure is given to the routine microarray users.
<ol>
	<li>Prepare 100 pg high quality RNA sample (RNA Integrity number (RIN) value at least &#62; 7.0) in a total volume of 10 - 11 &#181;L.</li>
	<li>Amplify the RNA samples with an amplification kit being able to work with pico-scale samples (as little as 100 pg). 
	NOTE: Recommended amplification kit information is available in Material List and follows the manufacturer&#39;s instructions without any modification.</li>
	<li>Use a fluorescence-based quantitation to evaluate the profile of the size range of the amplified aRNA.</li>
	<li>Use the spectrophotometer instrument (based on the absorbance at 260 and 280 nm) to measure the concentration of the amplified aRNA.</li>
	<li>Store the amplified aRNA at -80 &#176;C until ready for labeling.</li>
	<li>Use 2 &#181;g of amplified RNA for fluorescent labeling according to ULS aRNA labeling kit user guide. 
	NOTE: Since Cyanine 547 and Cyanine 647 dye are sensitive to photo-degradation and Cyanine 647 dye is easily degraded by ozone, the labeling procedure takes place in a minimal amount of light and under an ozone-free environment.</li>
	<li>Turn on the ozone box (Figure 1A) until the level of ozone is 0.001 ppm.</li>
	<li>Do the reaction setup inside the ozone box (Figure 1B) by adding 2 &#181;L of Cyanine 547 or Cyanine 647, 2 &#181;L of labeling buffer, and then adjust to a final volume of 20 &#181;L with RNase-free water under the safelight. Add darkroom filter to the light source.</li>
	<li>Mix gently and incubate the tubes in a thermocycler at 85 &#176;C for 15 min. Place samples on ice for at least 1 min. Spin down to collect content of tubes before proceeding with RNA extraction kit, except steps related to DNase treatment (from 1.9 to 1.11), to clean up the Cy-dye-labeled amplified RNA.</li>
	<li>Use the spectrophotometer instrument to measure and determine the concentration of labeled amplified RNA and keep the labeled aRNA at -80 &#176;C maximum for 3 days before use.</li>
</ol>
3. Microarray Hybridization and Washing
NOTE: The following important steps are adapted according to Two-color Microarray-based Gene Expression Analysis Protocol (version 6.5, May 2010).
<ol>
	<li>Add equal amount of 825 ng from each Cyanine 547- and Cyanine 647-labeled aRNA and mix with 25x fragmentation and 10x blocking buffers.</li>
	<li>Heat the mixture at 60 &#176;C for 15 min and cool on ice for 1 min.</li>
	<li>Add equal amount of 2x hybridization buffer and mix well.</li>
	<li>Centrifuge at 13,000 rpm, then the mixture is ready for hybridization.</li>
	<li>Load 100 &#181;L from the mixture onto the array slide and seal the slide inside the hybridization chamber.</li>
	<li>Load the assembled chamber into the hybridization oven for 17 h at 65 &#176;C rotating at 10 rpm in oven.</li>
	<li>Wash the arrays according to protocol with ozone protection treatment by dipping into stabilization and drying solution for 30 s.</li>
	<li>Remove the arrays from the solution and make sure the array surface is dry with no dust particles</li>
</ol>
4. Scanning Microarray Slide
<ol>
	<li>Keep the arrays in dark place and turn on scanner until it is ready to use (15 min wait time).</li>
	<li>Load the array barcode on left, into the scanner and start scanning. Do the spot analysis according to the software user guide and save the data as (gpr) file format.</li>
</ol>
5. Statistical Analysis
<ol>
	<li>Download the FlexArray software http://genomequebec.mcgill.ca/FlexArray/license.php Click on &#8220;import raw data&#8221; and load the gpr files into FlexArray.</li>
	<li>Perform the normalization and statistical analysis if necessary by following the instruction. Note: Calculate the threshold for positive spot selection in this study as it was described previously22.</li>
	<li>Select the drop down manual from the plot viewer of the FlexArray to view all the hybridized spots and background signals. Note: A similar picture of spiked in controls after hybridization to the microarray can be seen in previous study8.</li>
	<li>Select a Loess normalization within array following by a quantile between arrays normalization process from the FlexArray drop down manual accordingly.</li>
	<li>Export all the normalized data from FlexArray into an Excel file for further usage.</li>
</ol>
6. Bioinformatics Analysis
The original annotation of the probe sequences from Bovine Embryo-specific Transcripts (BESTv1) microarray has been described previously8. In present study, 20,403 unique Gene Symbols (GS) were obtained (<a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53754/Supplement_file_1.xlsx">Supplement File1</a>) through the EmbryoGENE Laboratory Information Management System (LIMS) ELMA (http://elma.embryo-gene.ca). A list of 6,765 unique genes (<a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53754/Supplement_file_2.xlsx">Supplement File2</a>) was selected and corresponded to the all positive signals after microarray normalization process (Step 5.5) from bovine Day 7 embryos gene expression profile. Positive signal threshold was calculated based on pervious publication16 from the signal intensity of an A value.
<ol>
	<li>To perform functional analysis with PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System23, 24, open the following link (http://www.pantherdb.org/about.jsp).</li>
	<li>Upload a list of 6,765 embryo-specific GS to identify specific biological process during embryonic development using statistical overrepresentation test and use default setting of Bos taurus (all genes in database) as reference list25. 
	NOTE: This allows the identification of developmental-related processes from the GO terms that are statistically over- or under-expressed using a binomial test.</li>
	<li>Set P-value threshold at &#60; 0.05 as software default. Data can be downloaded and exported into Excel file for further selection16.</li>
</ol>

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The authors have nothing to disclose.

The first problem to perform microarray analysis using Day 7 bovine embryos is not getting sufficient amounts of high quality RNA for studying gene expression. Traditional phenol/chloroform RNA extraction and ethanol precipitation method is not recommended for Day 7 embryos, resulting in low yield and possible leftover phenol inhibiting RNA amplification reaction. Instead, a standard column-based method is better to isolate total RNA and then elute the RNA with minimum elution buffer to increase the concentration. An ave...

Early embryonic loss in high-producing dairy cows is one of the main challenges in the dairy industry1,2. Bovine has become an interesting model to study human preimplantation embryo development due to their similar developmental process3,4. However, more research is required to have better understanding about genes involved in bovine early embryonic development.
After twenty years since the first microarray technology developed in 19955, the development of more sophisticated probe fabrication technology reduced printing errors and variability of array chips within and between different microarray platforms6. Improved microarray technology resulted in a widely application of this technology in clinical research7 and more recently, in early embryo quality assessment8.
The large amount of required material for microarray technology is the main reason why the microarray technology initially failed to enter a number of research fields such as early embryonic development. More recently, RNA amplification methods have been improved to linearly amplify RNA up to micrograms level from sub-nanogram starting RNA material9. There are several commercial RNA amplification kits available on the market; however, the more popular well developed kits are related to Ribo-Single Primer Isothermal Amplification10 and T7 promoter driven11 methods. The most popular antisense RNA amplification uses in vitro transcription with an oligo dT primer linking to a T7 promoter at 5&#39; end12. This technology allows maintaining the most of representative anti-sense transcripts after linear amplification for arrays hybridization13. This method has been adapted to amplify picogram level of total RNA extracted from bovine embryo8.
Universal Linkage System (ULS) is the labeling method which directly incorporates the DNA or amplified RNA with platinum-linked fluorescent dye either Cyanine 547 or Cyanine 647, by forming a coordinative bond on N7 position of guanine14. This method was adapted in embryos research to generate more stable amplified aRNA without modification compared to the aminoallyl modified aRNA generated by enzymatic method15. Both single dye and two dyes labeling methods have been adapted using Universal Linkage System in microarray. A large microarray comparisons study was that there is a good correlation of data quality between the one- and two-color array platforms6.
Recently, both T7 promoter driven antisense RNA amplification and ULS labeling methods have been developed to provide a more reliable protocol to generate a sufficient amount of high quality labeled aRNA materials for microarray hybridization8,16. Therefore, this study provides a protocol to demonstrate some of the important steps from RNA extraction to data analysis involved in two-color microarray using Day 7 bovine embryos as an example.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set (50)</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus RiboAmp HS PLUS Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0505</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instruments</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Screen Tape</td>
			<td>Agilent Technologies</td>
			<td>5067-5576</td>
			<td></td>
		</tr>
		<tr>
			<td>ULS Fluorescent Labeling Kit</td>
			<td>Kreatech Diagnostics</td>
			<td>EA-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gene Expression Microarrays</td>
			<td>Agilent Technologies</td>
			<td>G2514F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Agilent Gene Expression wash buffer 1</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5325</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Gene Expression wash buffer 2</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5326</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Hi-RPM Hybridization buffer</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5190-0403</td>
			<td></td>
		</tr>
		<tr>
			<td>25X Fragment buffer</td>
			<td>Agilent Technologies</td>
			<td>Part #5185-5974</td>
			<td></td>
		</tr>
		<tr>
			<td>10X GE Blocking Agent</td>
			<td>Agilent Technologies</td>
			<td>Part #5188-5281</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and drying solution</td>
			<td>Agilent Technologies</td>
			<td>&#160;Part #5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Gasket slides enabled by Agilent SureHyb techonolgy</td>
			<td>Agilent Technologies</td>
			<td>G2524-60012</td>
			<td>Pack of 20 gasket slides, 4 microarrays/slide</td>
		</tr>
		<tr>
			<td>Two-Color RNA Spike-In Kit</td>
			<td>Agilent Technologies</td>
			<td>&#160;Cat# 5188-5279</td>
			<td></td>
		</tr>
		<tr>
			<td>GenePix 4000B array scanner</td>
			<td>Molecular Devices</td>
			<td>GENEPIX 4000B-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Ozone Free Box</td>
			<td>BioTray</td>
			<td>OFB_100x200</td>
			<td></td>
		</tr>
		<tr>
			<td>GAL file</td>
			<td>Agilent Technologies</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A representative result of total RNA and amplified aRNA from Day 7 bovine embryos is shown in Figure 3 and summarized in Table 1.
RNA integrity and profile can be assessed after RNA extraction. Quality assessment of RNA could be done by bioanalyzer instrument (Figure 3A) and only those samples with RIN value higher than 7.0 are qualified to be used for amplification (Tab...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

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.

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 ...

transcriptome-profiling-vivo-produced-bovine-pre-implantation-embryos

Research

JoVE Journal

Developmental Biology

7.7K Views.  University of Alberta. 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.

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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...