same-day genotyping of isolated gastrulating murine embryos

Isolering och genotypning av gastrulaterande musembryo för encellssekvensering

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.

Författare i fokus: En pipeline för att analysera härstamningsspecifika muterade embryon med encellsupplösning

Encells-RNA-sekvensering av muterade hela musembryon: från epiblasten till slutet av gastrulationen

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.

Genotypning samma dag av isolerade gastrulerande murina embryon

same-day-genotyping-of-isolated-gastrulating-murine-embryos

Research

JoVE Journal

Developmental Biology

Same-Day Genotyping of Isolated Gastrulating Murine Embryos

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.

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 ...

Isolation of Murine Embryos During Gastrulation for Single-Cell RNA Sequencing

Prior to starting the murine embryo isolation, clean the area thoroughly with 70%ethanol. Ensure all dissection tools are washed and sterilized. Obtain five milliliters of sterile DMEM containing 10%FBS, and 20 milliliters of DPBS and place them on ice.Next to perform the embryo isolation, using a stereo microscope with a transmitted light stage and camera, place the properly euthanized pregnant dam on its back and sterilize the area near the vaginal opening with ethanol. Using dissection scissors, and tweezers, lift the skinfold near the vaginal opening and make a small V-shaped cut slowly revealing the uterus of the pregnant dam. Then dissect out the uterine horn of the dam by holding one end of it with tweezers and cutting along it.Making sure to remove the cervix. Place the uterine horn into a 10 centimeter Petri dish containing DPBS on ice. With dissection scissors, and tweezers, remove the uterine muscle along the implantation sites and cut each implantation site or pearls containing the decidual swellings inside.Place it into fresh DPBS in a six centimeter Petri dish on ice. Take one implantation site. Place it in a new six centimeter Petri dish on top of the stereo microscope stage, and add 500 microliters of DPBS to it.Adjust the focus of the microscope and the light source. Hold down the implantation site with one set of tweezers in one hand, and with the other hand slowly insert another pair of forceps into the end of the implantation site cut from the uterine horn, gradually revealing the deciduous swelling. To reveal the embryo, hold the anti mistrial end of the isolated decidual swelling with one pair of forceps and with the other pair, slowly make a horizontal cut about one quarter of the size of the decidual swelling from the mistrial end.Now with both forceps slowly push from the anti mistrial end of the decidual swelling until the embryo pops out from the freshly cut mistrial end. Once the embryo is revealed, proceed to remove any extra embryonic tissues attached. If the parietal endodermal sac and ecto placental cone do not spontaneously come off the embryo, use a pair of forceps to remove them along with any associated maternal blood.Then while holding down the embryo with one pair of forceps, slowly peel the visceral yolk sack from the embryo using another pair of forceps. Locate the parietal endodermal sac and ectoplacental cone. Using AP 20 pipette, transfer the yolk sac with no more than 10 microliters of DPBS from the dish into an eight strip PCR tube on ice.Take bright field pictures of freshly isolated embryos to ensure the staging of the litter mates is similar. With a P200 pipette slowly transfer the embryo with 50 microliters of DMEM containing 10%FBS into a 1.5 milliliter tube kept on ice. Repeat the same for all decidual swelling, using clean tools and new plastics for each isolation.

Isolering av murina embryon under gastrulation för encells-RNA-sekvensering

After isolating the visceral yolk sac from the mouse embryo, proceed to digest each yolk sac in an eight-strip polymerase chain reaction or PCR tube. Using a P20 pipette, add 19.3 microliters of PCR template DNA lysis buffer, and 0.7 microliters of 0.2 micrograms per milliliter proteinase K to each yolk sac sample. Vortex the sample for 10 seconds.And using a mini centrifuge with a strip adapter, spin down the samples at 1000 G for 10 seconds. Place the eight-strip PCR tube in an 85 degree Celsius heat block for 45 minutes while vortexing for five seconds every five minutes. After 45 minutes, spin the tube strip down again at 1000 G for 10 seconds before proceeding with PCR for desired genetic identification.To perform the PCR reaction for Cre genotyping, prepare a PCR master mix containing the displayed components per eight strip PCR tube for each yolk sac. Proceed to run the PCR thermal cycle amplification program using the displayed cycle. Then run the PCR products on a 1%agarose gel to draw genotyping conclusions.Store the remaining digested yolk sac samples in a minus 20 degree Celsius freezer for long-term storage. When the PCR product was separated on an agarose gel, expected fragment sizes for the LoxP and wild type alleles, as well as the Cre allele were seen.

Cell Dissociation of Isolated Gastrulating Murine Embryos for Single-Cell Sequencing

Proceed to perform the cell dissociation of isolated murine embryos once the genotypes have been confirmed. Using a P200 pipette, add 50 microliters of DMEM with 10%FBS to a new 1.5 milliliter tube. After pooling embryos with the same genotype into the new tube, place it on ice.Allow the pooled embryos to settle to the bottom of the tube, then wash the embryos using 50 microliters of DPBS and wait for them to settle before removing as much DPBS as possible without removing the embryos. Next, add 100 microliters of trypsin to the pooled embryos and incubate at 37 degrees Celsius in a heat block for five minutes. Gently flick the 1.5 milliliter tube every 30 seconds to help the cells dissociate.After five minutes, neutralize the trypsin with 300 microliters of DMEM containing 10%FBS. Centrifuge the embryos at 100 G for four minutes at room temperature. Resuspend the obtained small pellet in 40 microliters of DMEM with 10%FBS and place the tube on ice.Mix five microliters of the cell suspension with five microliters of trypan blue in a new 1.5 milliliter tube, and pipette the mixture up and down thoroughly. Transfer the mixture onto a slide to determine the cell number and viability using an automated cell counter. Finally, proceed to single cell partitioning using a microfluidic chip, following the manufacturer's protocol.