We acknowledge the Genomics Core at the Fox Chase Cancer Center and Dr. Johnathan Whetstine laboratory members Zach Gray, Madison Honer, and Benjamin Ferman for technical support for the sequencing experiments. We acknowledge laboratory members of Dr. Estaras and Alex Morris, a rotation graduate student who contributed to the initial analysis of the single-cell studies. This work is funded by the NIH grants R01HD106969 and R56HL163146 to Conchi Estaras. Additionally, Elizabeth Abraham was supported by T32 training grant 5T32HL091804-12.

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

<ol>
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M., Marikawa, Y., Moris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastruloids:+Pluripotent+stem+cell+models+of+mammalian+gastrulation+and+embryo+engineering.">Gastruloids: Pluripotent stem cell models of mammalian gastrulation and embryo engineering.</a> Dev Biol. 488, 35-46 (2022).</li><li>Kim, Y., Kim, I., Shin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+era+of+stem+cell+and+developmental+biology:+from+blastoids+to+synthetic+embryos+and+beyond.">A new era of stem cell and developmental biology: from blastoids to synthetic embryos and beyond.</a> Exp Mol Med. 55 (10), 2127-2137 (2023).</li><li>Pijuan-Sala, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+molecular+map+of+mouse+gastrulation+and+early+organogenesis.">A single-cell molecular map of mouse gastrulation and early organogenesis.</a> Nature. 566 (7745), 490-495 (2019).</li><li>Mittnenzweig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-embryo,+single-cell+time-resolved+model+for+mouse+gastrulation.">A single-embryo, single-cell time-resolved model for mouse gastrulation.</a> Cell. 184 (11), P2825-P2842 (2021).</li><li>Bardot, E. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+recording+of+mammalian+embryogenesis.">Molecular recording of mammalian embryogenesis.</a> Nature. 570 (7759), 77-82 (2019).</li><li>Mohammed, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+landscape+of+transcriptional+heterogeneity+and+cell+fate+decisions+during+mouse+early+gastrulation.">Single-cell landscape of transcriptional heterogeneity and cell fate decisions during mouse early gastrulation.</a> Cell Rep. 20 (5), 1215-1228 (2017).</li><li>Lescroart, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+earliest+step+of+cardiovascular+lineage+segregation+by+single-cell+RNA-seq.">Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq.</a> Science. 359 (6380), 1177-1181 (2018).</li><li>Scialdone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+early+mesoderm+diversification+through+single-cell+expression+profiling.">Resolving early mesoderm diversification through single-cell expression profiling.</a> Nature. 535 (7611), 289-293 (2016).</li><li>Chromium Next GEM single cell 3' reagent kits v3.1: User guide. 10X Genomics Available from: <a target="_blank" href="https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry">https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry</a> (2024)</li><li>NextSeq 1000/2000 Sequencing v2.0 Protocol. 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The authors have nothing to disclose.

A robust pipeline is presented in this paper for obtaining high-quality single-cell and nuclei suspensions from gastrulating mouse embryos, specifically designed to facilitate studies on mechanisms of cell-fate specification in early development. This method addresses a crucial gap in the field of gastrulation by optimizing the analysis of embryos requiring genotypes, such as sex or somatic genes. By utilizing genetic mutation mouse models and employing high-resolution single-cell sequencing on whole mouse embryos, this pipeline can further enhance the understanding of the gene expression profiles of the early mouse gastrula. This method demonstrates the feasibility of using a genetic mutation mouse model by a Cre recombinase system to obtain high-quality cells and nuclei at early developmental time points for single-cell omics. This method evolved through multiple attempts, during which samples did not meet the quality standards required for library preparation and sequencing. The explained methodology generates sufficient cells/nuclei from embryos younger than E8 through the optimization of three critical steps: (1) synchronized timed pregnancies to increase the number of embryos, (2) same-day genotyping to avoid freezing/thawing, and (3) assessing cell/nuclei viability to avoid sequencing of dying cells. This protocol delivers successful results with single-cell RNASeq, but the cell or nuclei suspensions obtained in this protocol can be processed in other sequencing platforms, where cell viability is the limiting factor, such as smart-seq, drop-seq, and cel-seq16.
At the E7 embryo, the number of cells can vary depending on the strain and mutant genotype. Typically, a E7 embryo can range from hundreds to a few thousand cells, and obtaining embryos with the desired genotype is challenging, with only 1 or 2 embryos per pregnant dam meeting the criteria. This protocol allows the capture of around 300 viable cells per E7 embryo for single-cell analysis. Attempts to increase embryo pool size by snap-freezing embryos from pregnant dams on different days proved unsuccessful, as cell viability was severely compromised after thawing, even with the addition of cryopreserving agents. To address this challenge, the breeding strategy and synchronization of pregnant dams was optimized. To increase the chances of multiple isolations, it is recommended to use female mice that have given birth 1-2 times before breeding for isolation, as they are more likely to have larger litter sizes. Monitoring the female&#39;s estrous cycle is crucial; mating is more likely to occur during the proestrus and estrus stages. If breeding difficulties arise, switching the breeding partners after four days will also help if no plug is produced.
It&#39;s important to note that this method has a limitation: it relies on observed plugs, and even if a plug is observed, it does not guarantee pregnancy but only indicates sexual activity. Therefore, increasing the number of observed plugs in a day will increase the probability of having more than one pregnant dam in a day and more positive genotypes. This protocol demonstrated the feasibility of using embryonic stages ranging from E7 to E7.75 as a proof of concept during gastrulation. However, this pipeline can be applied from E6.5 to E8 embryos. For E6.5 embryos, it is recommended to increase the number of synchronized pregnancies to obtain at least 7 embryos with the desired genotype to pool. Instead, for the E8 embryos, increase the amount of trypsin used to dissociate each embryo to ~ 40 &#181;L instead of 20 &#181;L. The goal is to obtain a cell/nuclei suspension in a concentration range of 700-1200 cells per &#181;l before proceeding with the partitioning.
Having good cell viability is essential for the success of single-cell sequencing. In the microfluidic chip design provided by the manufacturers, single cells are partitioned into gel beads-in-emulsion (GEMs) within a chip containing known barcoded gel beads11. However, a notable limitation of this process is that both high-quality and poor-quality single cells can be partitioned. Even with an adequate number of live cells (i.e., 1000 cells/&#181;L), if the viability of the suspension is low (i.e., 1000 cells alive and 1000 cells dead, resulting in 50% viability), the sequencing experiment will likely fail. For optimal results, it is recommended to aim for a viability of around 90%. If the cell viability falls between around 60%-89%, specific measures can be taken to enhance the experiment&#39;s viability. However, if the cell viability is less than 60%, it is strongly advised against continuing with the experiment. The reason for this is that the dying cells will be &#39;captured&#39; in the partitioning gel, and subsequent library preparation and quality controls will pass without noticeable issues. However, the actual sequencing experiment may completely fail, as most of the sequencing reads will map mitochondrial, ribosomal, or apoptosis genes, indicating signs of poor cell viability from the outset. Data presented in this paper illustrates a pipeline for single-cell sequencing of gastrulating mouse embryos with high-quality cells. This methodology can be applied for the understanding of many different genetic mutation mouse models during development.

Gastrulation is a fundamental process required for normal development. This rapid and dynamic process occurs when pluripotent cells transition into lineage-specific precursors that define how organs form. For years, gastrulation was long defined as the formation of three largely homogeneous populations: mesoderm, ectoderm, and endoderm. However, high-resolution technologies and an emerging number of embryonic stem cell models1,2 unveil unprecedented heterogeneity among the early germ layers3,4. This suggests that much more remains to be uncovered about the mechanisms regulating the distinct cell populations of the gastrula. Mouse embryonic development has been one of the best models to study early cell fate decisions during gastrulation3,5. Gastrulation in mice is rapid, as the entire process of gastrulation occurs within 48 h, from embryonic day E6.5 to E85.
Recent advancements in single-cell technologies have enabled detailed mapping of wild-type mouse embryonic development, providing a comprehensive overview of the cellular and molecular landscapes of embryos during gastrulation3,4,6,7,8. However, the analysis of mutant embryos at these stages is less common and often limited to FACS-sorted populations9,10. The scarce literature reflects the technical challenges associated with the manipulation and single-cell preparation of gastrulating embryos that require genotyping. Capturing the dynamic process of gastrulation can pose challenges due to its rapid nature, especially for understanding mutant embryos. The timing and synchronization of pregnancies are essential, as even slight differences between timed pregnancies can be misinterpreted as a developmental phenotype resulting from the mutant gene. This becomes particularly important when the mutant gene influences the process of gastrulation13,14. In this study, guidelines are established to obtain synchronized pregnancies through visualization of vaginal plugs (i.e., the mass of coagulated seminal fluid formed in the female&#39;s vagina after mating). Additionally, a strategy is designed to obtain robust single-cell data from mutant gastrulating embryos from E6.5 to E8. This strategy is devised to overcome constraints associated with the low number of embryos with the desired genotype per pregnancy and the decrease in viability caused by freezing-thawing embryos or cells.
This paper describes an optimized methodology from the establishment of timed pregnancies via vaginal plugs to the final sequencing of single cells/nuclei. This method explains how to increase the number of synchronized pregnancies to obtain a higher number of embryos with desired genotype, cell/nuclei isolations to improve the viability of the cells, and a same-day genotyping protocol. This manuscript also describes the process of embryo isolation at different gastrulation time points. The methodology helps to increase the number of final viable embryo cells/nuclei for sequencing, ensuring high-quality sequencing data. Therefore, this method will open the doors for single-cell studies of gastrulating embryos that require genotyping.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dish</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Reach Barrier Tip</td>
			<td>Genesee Scientific</td>
			<td>23-430</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L Reach Barrier Tip</td>
			<td>Genesee Scientific</td>
			<td>24-404</td>
			<td></td>
		</tr>
		<tr>
			<td>300 &#181;L Reach Barrier Tip</td>
			<td>Genesee Scientific</td>
			<td>24-415</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;m Reversible Strainer, small</td>
			<td>Stem Cell</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Genesee Scientific</td>
			<td>25-260</td>
			<td></td>
		</tr>
		<tr>
			<td>8-strip PCR tubes</td>
			<td>Genesee Scientific</td>
			<td>27-125U</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Apex Bioresearch Product</td>
			<td>20-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Scientific BSH300 MyBlock Mini Dry Bath</td>
			<td>Genesee</td>
			<td>31-437</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Scientific Z216-MK Z216MK Hermle Refrigerated Microcentrifuge</td>
			<td>Genesee</td>
			<td>33-759R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Controller</td>
			<td>10X</td>
			<td>PN-1000127</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; Reagent Kits v3.1</td>
			<td>10X</td>
			<td>PN-1000269</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAX2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Couning Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>D1000 Reagents</td>
			<td>Agilent</td>
			<td>5067-5583</td>
			<td></td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>ThermoFisher Scientific</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>DirectPCR yolk sac</td>
			<td>Viagen</td>
			<td>201-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher Scientific</td>
			<td>R0861</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tube 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>Dubecco&#39;s Modificiation of Eagle&#39;s Medium (DMEM, 1x)</td>
			<td>CORNING</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>GenClone</td>
			<td>25-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>V1401</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS M7000 Imaging System</td>
			<td>Invitrogen</td>
			<td>AMF7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq G2 Green Master Mix</td>
			<td>Promega</td>
			<td>M7823</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11049-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>ThermoFisher Scientific</td>
			<td>AC223211000</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAmp Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq 1000/2000 P2 Reagents (100 Cycles) v3</td>
			<td>Illumina</td>
			<td>20046811</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq2000</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ 1000 Stereo Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nondiedt P40</td>
			<td>Sigma-Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>GenClone</td>
			<td>25-511</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Tube 8x Strip (401428)</td>
			<td>Agilent</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Tube Cap 8x Strip (401425)</td>
			<td>Agilent</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr>
			<td>Poseidon 31-511, HS24 Microcentrifuge, with 24 x 1.5/2.0 mL rotor, 1 Centrifuge/Unit</td>
			<td>Genesee</td>
			<td>31-511</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA Quantification Assay Kits</td>
			<td>Invitrogen</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Flex 3</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>Fisher Scientific</td>
			<td>12-141-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape Station Loading tips</td>
			<td>Agilent</td>
			<td>5067-5598</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapestation 4150</td>
			<td>Agilent</td>
			<td>G2992AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL (Ph7)</td>
			<td>Quality Biological</td>
			<td>351-007-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain 0.4%</td>
			<td>Theromo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple Express</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bio-Rad</td>
			<td>1662404</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer IKA MS3 with 96-well sample plate adapter</td>
			<td>IKA</td>
			<td>3617000</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell rna sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

The methodology designed in this paper is specifically intended to enhance the preparation of embryo samples for single-cell omics from E6.5 to E8. This robust pipeline consists of five major steps: synchronized timed pregnancies, embryo isolations, same-day genotyping, cell dissociation, and assessment of cell viability (Figure 1A). While the presented data focuses on time points from E7 to E7.5, it can be applied to embryos up to E8 (Figure 1B)&#160;with small variations in the procedure (referred to notes throughout the protocol). Synchronized timed pregnancies were achieved by placing two female mice into a cage with a male mouse. Vaginal plugs were checked every morning before 10 AM, and if a plug was observed, it was considered E0.5 by noon of that day (Figure 2A). In this study, optimal conditions for plugs required a female mouse in the estrus or proestrus stage to be paired with a male with a history of successfully placing several plugs during prior breeding (Figure 2B). Only obvious plugs were considered for embryo isolations (Figure 2C).
The developmental timing was estimated following the timetable in Figure 2A. For E7.5, embryo dissection started at 12 PM on the 7th day following the day of the detection of a plug. Figure 3 exemplifies a successful embryo isolation at E7.5. After the pregnant dam was euthanized, the uterine horn was dissected, the decidual swelling was individually cut, revealing the embryo, and the yolk sack was isolated for genotyping (Figure 3).
Same-day genotyping was performed within 3 h of embryo isolation following the steps in Figure 4A. The embryos were kept on ice during the process of genotyping to preserve their integrity. Do not freeze the embryos, as it will decrease the number of viable cells per embryo. Figure 4B,C show the visceral yolk sack, the parietal endodermal sac, and the ectoplacental cone with associated maternal blood. The visceral yolk sac was utilized for genotyping (Figure 4C). After digesting the yolk sac, the PCR mix was prepared, and the PCR reaction was run. The resulting PCR product was then separated on an agarose gel. Figure 4D shows expected fragment sizes for the LoxP and wild-type alleles (597 bp and 498 bp, respectively) and the Cre allele (650 bp). In Figure 4E, an example of yolk sac genotyping from 6 embryos obtained from breeding pairs carrying Cre and LoxP (flox) alleles is presented. The gels depict the PCR products of the Cre and LoxP alleles in the 6 embryos analyzed. Embryos number 2 and number 5 carry 2 flox alleles and 1 cre allele; therefore, they are considered conditional KO embryos (Figure 4E, red dots). Embryos 1 and 3 have 2 flox alleles but are negative for Cre; therefore, they are considered flox controls. Embryo 4 has 2 flox alleles and a faint band for the Cre allele, resulting in an &#34;unclear&#34; genotype. This embryo was not further processed (alternatively, the experimentalist may consider repeating the genotyping or conducting a secondary validation of the conditional KO using cells expressing Cre). It is important to note that the Cre driver used in this experiment is not expressed in the visceral yolk sac15; hence, the LoxP alleles do not appear shifted on the gel of Cre-positive embryos compared to the Cre-negative ones.
Cell count and good viability are required for a successful single-cell experiment. Suspensions with low cell viability, a high percentage of dead cells, clumping, or significant debris are unsuitable for further processing. Optimal conditions are for 700-1200 live cells per microliter and &#62;90% viability. Figure 5A presents a panel illustrating both good and sub-optimal cell viability. The same criteria can also be applied for nuclei isolation, as shown in Figure 5B. However, it is crucial to note that the evaluation of trypan blue differs from cells and nuclei: viable cells do not incorporate trypan blue, while viable nuclei do. If cells/nuclei suspension is optimal (&#62;90% viability), proceed with single-cell partitioning using a microfluidic chip following manufacturer&#39;s procedures11. Troubleshooting options are provided for suspensions where cell/nuclei viability is between 60%-89%, as depicted in Figure 5C. If viability, regardless of the total number of cells, falls below 60%, consider halting the experiment.
The entire pipeline from the culling of the pregnant dam to library construction takes a total of 8 hours on the same day (i.e., protocol steps 2 to 6 must be performed on the same day). Following the procedures outlined by the single-cell manufacturers&#39; procedures11, library constructions were prepared for single-cell RNA sequencing using cells obtained from E7 embryos, with cell viability ranging from sub-optimal to optimal conditions, aiming for an estimated target recovery of 2000 cells (Figure 6). Figure 6A depicts a representative fragment size distribution of scRNAseq libraries for both sub-optimal and optimal conditions, indicating that cell viability does not significantly affect the entire single-cell partitioning process. The fragment size distribution ranged between 400-500 bp. This indicates that sub-optimal conditions do not affect the process of library preparation. Figure 6B shows the outcomes of both successful and sub-optimal single-cell RNAseq experiments. Following sequencing, quality control checks were conducted on samples and observed that, in cases where cell viability was sub-optimal, only 10% of cells were successfully sequenced. In contrast, optimal samples exhibited a higher percentage, with 91% of the total cells being sequenced. This is further proven by barcode plots, indicating that the sub-optimal conditions have larger background noise compared to optimal conditions. Clustering analysis was performed for both experiments and revealed 9 clusters in the optimal conditions and 4 in the sub-optimal. Annotation of the clusters using known markers3 revealed expected cell -types in the E7 embryos, including epiblast and primitive streak (Figure 6B). Cluster annotations in the suboptimal experiment were not possible due to the lack of enrichment in known markers for each cluster. This highlights the importance of high-quality cells required for the proper representation of data during these stages of development.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66866/66866fig1v2.jpg" /> 
Figure 1: Optimization of&#160;gastrulating whole mouse embryos for single-cell RNA sequencing. (A) Workflow schematic for obtaining high-quality cells and/or nuclei from gastrulating embryos. (B)&#160;Representative bright field images and adapted scheme3 of mouse embryos during gastrulation from E7 to E8. Scale bar: 125 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66866/66866fig2v2.jpg" /> 
Figure 2: Strategy for efficient synchronized timed pregnancies for embryo isolations during gastrulation. (A)&#160;Schematic diagram indicating the pipeline for timed pregnancies and a timetable describing the times from plug detection to embryo isolation for analysis of specific gastrulation phases. (B) Representative images of the phases of the estrus cycle for female mice. The phases most receptive to breeding are indicated. (C)&#160;Schematic diagram illustrating how to check for vaginal plugs and examples of vaginal openings without a plug, a partial plug, or a good plug to consider for embryo isolations. The vaginal openings are zoomed in below each image. The plug (coagulated semen in the vagina opening) is highlighted with a red arrow pointing to it. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66866/66866fig03.jpg" /> 
Figure 3: Dissection and genotyping of E7.5 embryos for single-cell RNA sequencing. Schematic diagrams and images of the process of isolating E7.5 embryos and dissecting the visceral yolk sac. The yellow arrow indicates the uterus of the pregnant dam, and the yellow dashed circle outlines the location of the embryo. Dashed lines represent the areas that were cut during dissection. &#34;M&#34; denotes the mesometrial end, while &#34;AM&#34; denotes the anti-mesometrial end. Scale bars in stereoscope images are 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66866/66866fig04.jpg" /> 
Figure 4: Same-day genotyping of yolk sacs from gastrulating mouse embryos. (A)&#160;Workflow schematic for same-day genotyping of yolk sacs. (B)&#160;Representative image of the parietal endodermal sac and ectoplacental cone with associated maternal blood. (C)&#160;Visceral yolk sac in E7.5 embryos highlighted by the dashed line. Scale bar: 200 &#181;m. (D)&#160;Representative gels of wild type (+/+), heterozygous (Flox/+), and homozygous flox (Flox/Flox), and Cre genotyping (Mesp1cre15) with observed DNA fragment sizes. (E) Same-day genotyping results for Cre and flox alleles from yolk sacs of 6 embryos (1-6) are provided. The two embryos with flox/flox and Cre negative (-) genotyping are flox controls (blue dots), while those with flox/flox and Cre positive (+) genotyping are conditional KOs (red dots). Embryo 4 is not optimal due to the faint Cre band (flox/flox and Cre-unclear) and is consequently excluded from further processing. Embryos with the same genotype are pooled and processed for single-cell RNA sequencing. Note that the presence of the Cre allele in the yolk sac does not affect the size of the flox bands , as the Cre used (Mesp1cre) is not expressed in the yolk sac. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66866/66866fig05.jpg" /> 
Figure 5: Assessment of cell quality and nuclei viability. (A) Representative images of optimal and sub-optimal cell viability conditions from E7.5 embryos. (B)&#160;Representative images of optimal and sub-optimal nuclei viability conditions from E8 embryos (C)&#160;Troubleshooting scheme indicating potential solutions to help increase the viability of cells before starting single-cell partitioning. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66866/66866fig06.jpg" /> 
Figure 6: Single-cell RNA sequencing of E7 mouse embryos. (A) Representative trace of fragment size distribution for single-cell RNA sequencing libraries from E7 mouse embryos for both sub-optimal and optimal conditions of cell viability, with the main peak near 400-500 bp. Note that the traces are similar between these conditions, indicating that the cell viability does not affect the quality controls of the library. (B) Analysis of single-cell RNA sequencing outcomes for E7 mouse embryos under both sub-optimal and optimal conditions showing observed target recovery, barcode ranking, and clustering of cell types through uniform manifold approximation and projection (UMAP) distribution. <a href="https://www.jove.com/files/ftp_upload/66866/66866fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Lysis Buffer</td>
		</tr>
		<tr>
			<td>Reagent Name</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
			<td>Total Volume 1 mL</td>
		</tr>
		<tr>
			<td>Tris-HCL (Ph7)</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 M</td>
			<td>10 mM</td>
			<td>2 &#181;L</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>0.1 M</td>
			<td>3 mM</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>20%</td>
			<td>0.10%</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Nondiedt P40</td>
			<td>10%</td>
			<td>0.10%</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>5%</td>
			<td>0.01%</td>
			<td>2 &#181;L</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>&#160;1 M</td>
			<td>1 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>40 U/&#181;L</td>
			<td>1 U/&#181;L</td>
			<td>25 &#181;L</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>--</td>
			<td>--</td>
			<td>924 &#181;L</td>
		</tr>
		<tr>
			<td colspan="4">Wash Buffer</td>
		</tr>
		<tr>
			<td>Reagent Name</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
			<td>Total Volume 1 mL</td>
		</tr>
		<tr>
			<td>BSA in PBS</td>
			<td>10%</td>
			<td>1%</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>--</td>
			<td>--</td>
			<td>900 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Nuclei isolation lysis buffer and wash buffer composition.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

single-cell-rna-sequencing-mutant-whole-mouse-embryos-from-epiblast

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

673 Views.  Temple University, Lewis Katz School of Medicine. This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei. 

Video: Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

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 ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...