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.

This protocol and all animal experiments described were formally approved and in accordance with institutional guidelines established by the Temple University Institutional Animal Care and Use Committee, which follows the Association for Assessment and Accreditation of Laboratory Animal Care international guidelines. All mice described were on the C57/BL6N background strain. No animal health concerns were observed in these studies.
1. Breeding colony and timed pregnancies
<o...

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

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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. S., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+gastrulation:+Coordination+of+tissue+patterning,+specification+and+diversification+of+cell+fate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-omics+profiling+of+mouse+gastrulation+at+single-cell+resolution.">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/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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Q., 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=TET-mediated+DNA+demethylation+controls+gastrulation+by+regulating+Lefty-Nodal+signalling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=editing-mediated+one-step+inactivation+of+the+Dnmt+gene+family+reveals+critical+roles+of+DNA+methylation+during+mouse+gastrulation.">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><li>Saga, Y., 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=MesP1+is+expressed+in+the+heart+precursor+cells+and+required+for+the+formation+of+a+single+heart+tube.">MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.</a> Development. 126 (15), 3437-3447 (1999).</li><li>Ziegenhain, C., 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=Comparative+analysis+of+single-cell+RNA+sequencing+methods.">Comparative analysis of single-cell RNA sequencing methods.</a> Mol Cell. 65 (4), 631-643 (2017).</li></ol>

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 ...

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.

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

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

We focus on deciphering the mechanisms controlling early cell fate decisions during embryonic stem cell differentiation, both in vitro and in mouse models. Complex regulatory networks guide differentiation during development, and we investigate how the interplay of signaling pathways, transcription factors, and epigenetic regulators promote different cell fates. Studying the dynamic and rapid process of gastrulation in mice is technically challenging due to the small number of cells per embryo and the limited number of embryos with the desired genotype per litter.While literature extensively shows the study of single cells using wild type or fluorescent-activated cell sorted populations of embryos, a comprehensive analysis of embryos with lineage mutations is still limited. Our protocol offers the possibility of generating single-cell omic datasets from gastrulating embryos harboring lineage mutations. It will help scientists with none or limited mouse handling experience or who wanna learn early embryo manipulation or single-cell approaches.Our protocol will help answer new scientific questions in gastrulation and early organogenesis, including heart development. We provide a pipeline to analyze lineage-specific mutant embryos at single-cell resolution, which will facilitate analysis of the role of a specific genes in lineage commitment. We hope to use this methodology to understand the regulatory mechanisms of cell fate decisions during the rapid and dynamic process of gastrulation.

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...

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 ...