Name: Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation (Video) | JoVE Journal
Uploaded: 2024-06-14T14:50:00.000+00:00
Duration: 9 min 14 s
Description: Learn single-cell RNA sequencing of gastrulating mouse embryos to study early cell fate decisions and lineage mutations. This protocol enables comprehensive analysis of embryonic development from epiblast to gastrulation end.

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5 minutes

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><li>Arias, A. M., Marikawa, Y., Moris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gastruloids%3A+Pluripotent+stem+cell+models+of+mammalian+gastrulation+and+embryo+engineering%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((A+new+era+of+stem+cell+and+developmental+biology%3A+from+blastoids+to+synthetic+embryos+and+beyond%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">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>
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<li>Mittnenzweig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-embryo%2C+single-cell+time-resolved+model+for+mouse+gastrulation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A single-embryo, single-cell time-resolved model for mouse gastrulation.</a> Cell. 184 (11), E22 P2825-P2842 (2021).</li>
<li>Bardot, E. S., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+gastrulation%3A+Coordination+of+tissue+patterning%2C+specification+and+diversification+of+cell+fate%5BTitle%5D)+AND+%22Mech+Dev%22%5BJournal%5D)">Mouse gastrulation: Coordination of tissue patterning, specification and diversification of cell fate.</a> Mech Dev. 163, 103617(2020).</li>
<li>Argelaguet, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-omics+profiling+of+mouse+gastrulation+at+single-cell+resolution%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Multi-omics profiling of mouse gastrulation at single-cell resolution.</a> Nature. 576 (7787), 487-491 (2019).</li>
<li>Chan, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+recording+of+mammalian+embryogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Single-cell+landscape+of+transcriptional+heterogeneity+and+cell+fate+decisions+during+mouse+early+gastrulation%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">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/pubmed?term=((Defining+the+earliest+step+of+cardiovascular+lineage+segregation+by+single-cell+RNA-seq%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq.</a> Science. 359 (6380), 1177-1181 (2018).</li>
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<li>Dai, H. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TET-mediated+DNA+demethylation+controls+gastrulation+by+regulating+Lefty-Nodal+signalling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling.</a> Nature. 538, 528-532 (2016).</li>
<li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((editing-mediated+one-step+inactivation+of+the+Dnmt+gene+family+reveals+critical+roles+of+DNA+methylation+during+mouse+gastrulation%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation.</a> Nat Commun. 14 (1), 2922(2023).</li>
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</ol>

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><tbody><tr><td>10 cm Petri dish</td><td>Genesee Scientific</td><td>25-202</td><td></td></tr><tr><td>1000 &amp;micro;L Reach Barrier Tip</td><td>Genesee Scientific</td><td>23-430</td><td></td></tr><tr><td>20 &amp;micro;L Reach Barrier Tip</td><td>Genesee Scientific</td><td>24-404</td><td></td></tr><tr><td>300 &amp;micro;L Reach Barrier Tip</td><td>Genesee Scientific</td><td>24-415</td><td></td></tr><tr><td>37 &amp;micro;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&#039; 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&#039;s Modificiation of Eagle&#039;s Medium (DMEM, 1x)</td><td>CORNING</td><td>10-013-CV</td><td></td></tr><tr><td>Dulbecco&#039;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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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

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) 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 "unclear" 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 &gt;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 (&gt;90% viability), proceed with single-cell partitioning using a microfluidic chip following manufacturer'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' 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="Embryo genotyping flowchart and gastrulation timeline diagram for single-cell microfluidic analysis." class="xfigimg" src="/files/ftp_upload/66866/66866fig1v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Optimization of gastrulating whole mouse embryos for single-cell RNA sequencing. (A) Workflow schematic for obtaining high-quality cells and/or nuclei from gastrulating embryos. (B) Representative bright field images and adapted scheme3 of mouse embryos during gastrulation from E7 to E8. Scale bar: 125 µm. <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="Mouse breeding timeline and receptivity stages; vaginal plug detection; embryo isolation guide." class="xfigimg" src="/files/ftp_upload/66866/66866fig2v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Strategy for efficient synchronized timed pregnancies for embryo isolations during gastrulation. (A) 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) 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="Embryo dissection diagram, showing step-by-step uterine extraction and embryo removal." class="xfigimg" src="/files/ftp_upload/66866/66866fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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. "M" denotes the mesometrial end, while "AM" denotes the anti-mesometrial end. Scale bars in stereoscope images are 400 µm. <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="Yolk sac isolation and PCR process, flowchart, embryo images, genotyping gel results, DNA analysis." class="xfigimg" src="/files/ftp_upload/66866/66866fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Same-day genotyping of yolk sacs from gastrulating mouse embryos. (A) Workflow schematic for same-day genotyping of yolk sacs. (B) Representative image of the parietal endodermal sac and ectoplacental cone with associated maternal blood. (C) Visceral yolk sac in E7.5 embryos highlighted by the dashed line. Scale bar: 200 µm. (D) 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="Cell viability analysis, microscopy images, and troubleshooting flowchart for microfluidic protocol." class="xfigimg" src="/files/ftp_upload/66866/66866fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
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) Representative images of optimal and sub-optimal nuclei viability conditions from E8 embryos (C) 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="Cell viability analysis; electrophoresis gel, histogram, barcode rank plot, UMAP clustering results." class="xfigimg" src="/files/ftp_upload/66866/66866fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
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 µL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 M</td>
			<td>10 mM</td>
			<td>2 µL</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>0.1 M</td>
			<td>3 mM</td>
			<td>3 µL</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>20%</td>
			<td>0.10%</td>
			<td>5 µL</td>
		</tr>
		<tr>
			<td>Nondiedt P40</td>
			<td>10%</td>
			<td>0.10%</td>
			<td>10 µL</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>5%</td>
			<td>0.01%</td>
			<td>2 µL</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td> 1 M</td>
			<td>1 mM</td>
			<td>1 µL</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>40 U/µL</td>
			<td>1 U/µL</td>
			<td>25 µL</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>--</td>
			<td>--</td>
			<td>924 µL</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 µL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>--</td>
			<td>--</td>
			<td>900 µL</td>
		</tr>
	</tbody>
</table>
Table 1: Nuclei isolation lysis buffer and wash buffer composition.

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

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

Research

JoVE Journal

Developmental Biology

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

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

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Here, we describe a protocol for generating maternal mutant that couples a stable zpc:cas9 knock-in line with Tol2-mediated delivery of sgRNA expression cassettes.

Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

This protocol provides a streamlined computational pipeline for quantifying nascent enhancer transcripts. By integrating chromatin accessibility, chromatin feature, and transcriptional data, it enables accurate detection and strand-specific analysis of enhancer activity in complex intragenic regions, while remaining accessible to researchers without extensive bioinformatics training.

A Computational Pipeline for Intergenic/Intragenic Enhancer RNA Quantification in Mouse Embryonic Stem Cells

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

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.

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

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.

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

Learn single-cell RNA sequencing of gastrulating mouse embryos to study early cell fate decisions and lineage mutations. This protocol enables comprehensive analysis of embryonic development from epiblast to gastrulation end.

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

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