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>
<li>Pijuan-Sala, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-cell+molecular+map+of+mouse+gastrulation+and+early+organogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/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>
<li>Scialdone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolving+early+mesoderm+diversification+through+single-cell+expression+profiling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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. Illumina. , Available from: 
 <a target="_blank" href="https://support-docs.illumina.com/SW/ClarityLIMS-INT/Content/SW/ClarityLIMS/Integrations/NextSeq10002000Intv20Config.htm">https://support-docs.illumina.com/SW/ClarityLIMS-INT/Content/SW/ClarityLIMS/Integrations/NextSeq10002000Intv20Config.htm</a> (2024).</li>
<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>
<li>Saga, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MesP1+is+expressed+in+the+heart+precursor+cells+and+required+for+the+formation+of+a+single+heart+tube%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+single-cell+RNA+sequencing+methods%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Comparative analysis of single-cell RNA sequencing methods.</a> Mol Cell. 65 (4), 631-643 (2017).</li>
</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 ...

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

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...

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

Research

JoVE Journal

Developmental Biology

775 Views.  Temple University, Lewis Katz School of Medicine. We richten ons op het ontcijferen van de mechanismen die vroege beslissingen over het lot van cellen bepalen tijdens embryonale stamceldifferentiatie, zowel in vitro als in muismodellen. Complexe regulerende netwerken begeleiden differentiatie tijdens ontwikkeling, en we onderzoeken hoe het samenspel van signaalroutes, transcriptiefactoren en epigenetische regulatoren verschillende celloten bevordert. Het bestuderen van het dynamische en snelle proces van gas...

Video: Author Spotlight: een pijplijn om afstammingsspecifieke mutante embryo's te analyseren met een resolutie van één cel

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

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

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.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

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 ...

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

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

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.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

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

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

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

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;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.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

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

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

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

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

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.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

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

Samenvatting

Meer video's verkennen