Isolating Single Cells from Xenopus Early Embryos and Sorting Them by Size

Summary

We describe a method for isolating single cells from Xenopus laevis early embryos and sorting them by cell size.

Abstract

Early embryogenesis requires an exponential decrease in cell size, while the embryo remains constant in size at the earliest stages of development. The functional importance and the mechanisms by which cell size impacts embryo development are still largely understudied. The Xenopus early embryo provides a unique system to address these major gaps. Compared with other model organisms, the Xenopus early embryo is extremely large and contains a cell-size gradient, from small cells at the animal pole to large cells at the vegetal pole, across the entire embryo at stages around the mid-blastula transition. Here, we describe a protocol for dissociating single cells from Xenopus early embryos and sorting them by size using cell strainers with varying mesh pore sizes. As determined by imaging and size measurements, the sorted cells are within the expected size range and can be used for live-cell analysis or sequencing, or fixed for imaging. This system will shed new light on revealing size-dependent regulatory mechanisms during early development.

Introduction

Cell size change is a common feature of early embryogenesis, when the fertilized egg undergoes multiple rounds of cleavage, generating an increasing number of cells. While much is known about the role of cell size as a major parameter controlling cellular functions^1,2,3, how cell size impacts early development is less known. Recent work across various model embryonic systems suggests that cell size regulates zygotic genome activation (ZGA)⁴ and cell fate specification⁵, supporting an important role for cell size in developmental decisions^6,7.

The Xenopus early embryo is an important model for studying cell size-regulated developmental decision-making. Because the Xenopus early embryo is large (~1.2 mm in diameter), it is easy to manipulate its size physically, making it useful for assessing the direct consequences of cell-size change. For example, mini embryos of varying sizes can be generated by constriction of 1-cell embryos using hair loops^4,8, demonstrating dose-dependent ZGA⁴. Moreover, Xenopus early embryo contains a gradient of cell sizes along the animal-vegetal axis, with the smallest cells at the animal pole and the largest cells at the vegetal pole, demonstrating an over 100-fold difference in volume⁴. Importantly, by directly visualizing and quantifying nascent transcripts in single cells of whole-mount embryos, we identified a cell-size threshold that regulates ZGA⁴. Further, isolation of single cells from Xenopus early embryos and sorting them by size confirmed the importance of the cell size-threshold ZGA⁴.

Here we describe the detailed procedures for isolating single cells from Xenopus laevis early embryos and sorting them by size. Xenopus early embryos can be dissociated into individual cells by incubating them in Ca²⁺, Mg²⁺-free medium (CMFM)⁹, and these single cells can further be sorted using cell strainers with varying mesh pore sizes. The sorted cells can be purified and used in various applications, including live cell analysis, sequencing, or imaging assays.

Protocol

Animal work described here has been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Carolina.

1. Solution preparation

NOTE: Before starting the protocol, prepare the following solutions.

1. Prepare 20× MMR: Mix 100 mM HEPES, 2 mM EDTA, 2 M NaCl, 40 mM KCl, 20 mM MgCl₂, and 40 mM CaCl₂. Adjust pH to 7.8 with NaOH. Autoclave and store at room temperature.
2. Prepare Ca²⁺, Mg²⁺-free medium (CMFM): Mix 50.3 mM NaCl, 0.7 mM KCl, 9.2 mM Na₂HPO₄, 0.9 mM KH₂PO₄, 2.4 mM NaHCO₃, and 1.0 mM EDTA. Adjust pH to 7.4, if necessary. Autoclave and store at room temperature.
3. Prepare a 1.5% agarose gel in 60-mm Petri dish: Add 0.75 g of agarose to 50 mL of CMFM. Heat to dissolve the agarose in a microwave oven. Add 5-10 mL of agarose gel to coat a thin layer in a 60-mm Petri dish.
4. Prepare a 15% density gradient medium (wt/vol)/CMFM: Slowly add 15 g of density gradient medium into 100 mL of CMFM. To facilitate the dissolving, heat and stir on a plate until the density gradient medium is completely dissolved. Keep the solution at 4 °C until use.
5. Prepare 4% PFA/1× MEM: Mix 1 vial (10 mL) of 16% paraformaldehyde stock and 4 mL of 10× MEM (1000 mM MOPS pH 7.4, 20 mM EGTA, and 10 mM MgSO₄). Add ultrapure water to 40 mL.
   NOTE: To obtain Xenopus embryos, follow a standard protocol to perform in vitro fertilization (IVF) (for example, see Sive et al., 2007)¹⁰. The embryo should be in 0.1× MMR ready for use.

2. Dissociating early embryos into single cells

1. Add ~15 mL of the CMFM to a new 60-mm petri dish coated with 1.5% agarose gel. Use a plastic transfer pipette to transfer 10-100 embryos (depending on the experimental needs) into the dish. Put the dish under a stereomicroscope.
2. Use tweezers in both hands to carefully remove the vitellin membrane by catching it on the surface of the embryos with one tweezer and peeling it off with the other. Avoid damaging the embryos during the process.
3. Incubate the devitellined embryos in the CMFM for ~1 h at room temperature. Shake the dish very gently every 5 min to facilitate the dissociation of cells. Check regularly until no piles of blastomeres are seen (Figure 1).

3. Sorting the dissociated cells by size

1. During the blastomere dissociation, submerge the cell stainers (40 µm, 70 µm, and 100 µm) in individual Petri dishes filled with CMFM (Figure 2). Place the dishes in the order from left to right as indicated. Also include two more CMFM-filled dishes between the groups for brief washing of cell stainers during each sorting.
2. Gently swirl the dish containing the isolated single blastomeres to enrich them at the center of the dish. Carefully transfer the blastomeres into the 40 µm stainer in CMFM.
3. Gently shake the strainer a few times to let the blastomeres < 40 µm settle through the strainer. Then quickly move the 40 µm strainer to the CMFM-filled dish for brief washing. Carefully transfer the blastomeres in the 40 µm strainer, which are > 40 µm, into the 70 µm strainer.
4. Repeat the steps until the blastomeres are sorted in the 100 µm strainer.
5. Gently swirl each dish with sorted blastomeres to enrich them at the center of the dish. Carefully transfer the blastomeres (~4 mL) onto the top of 0.5 mL of 15% density gradient medium in a fluorescence-activated cell sorting (FACS) tube (Figure 3). Avoid mixing.
6. Let the tube sit for 10 min. The intact blastomeres will settle down to the bottom by gravity, separating from the yolk globules from the burst blastomeres during the sorting and transfer (Figure 3).
7. Depending on the purpose of downstream experiments, proceed with live cell analysis, freeze the blastomeres in liquid nitrogen for RNA work, or fix the blastomeres for immunostaining (Figure 3).

Results

Xenopus laevis embryos (n = 50) at Stage 9 were incubated in the CMFM medium to dissociate into single blastomeres following this protocol (see an example in Figure 1). The dissociated single blastomeres were sorted sequentially using the 40 µm, 70 µm, and 100 µm cell strainers as described above. The unsorted control mix and he sorted single blastomeres were fixed in 4% paraformaldehyde and imaged under a bright-field microscope (Figure 4A). As shown in Figure 4B,C, as compared with the control unsorted blastomeres, which contained a wide range of sizes, the sorted blastomeres showed a narrow range of sizes that fall into the respective expected size ranges: < 40 µm, 33 ± 5.3 µm; 40-70 µm, 55 ± 11 µm; > 70 µm, 91 ± 17 µm (Figure 4), suggesting successful sorting of them based on the sizes. The size-sorted cells can be used for further analysis, such as validating cell size-dependent zygotic genome activation in early embryogenesis⁴.

Figure 1: Example of dissociated blastomeres. Image shows blastomeres dissociated at stage 9. Note the variation in the blastomere size. Scale bar: 100 µm. Please click here to view a larger version of this figure.

Figure 2: Experimental setup for sorting isolated blastomeres by size using cell strainers. All petri dishes are coated with 1.5% agarose gel and filled with CMFM. The cell strainers with 40 µm, 70 µm, and 100 µm mesh pore-size cutoff, shown in different colors, are placed in order in individual dishes. Two dishes in between are used for brief washing after each sorting. Isolated single blastomeres are transferred sequentially through each stainer. The mixed blastomeres can thus be separated into four subgroups based on size: <40 µm, 40-70 µm, 70-100 µm, and >100 µm. Please click here to view a larger version of this figure.

Figure 3: Purifying sorted blastomeres for various applications. During the sorting, many blastomeres can be burst into yolk globules, contaminating the blastomere populations. The unsorted and sorted blastomeres are transferred onto 0.5 mL of 15% density gradient medium in a FACS tube. After 10 min, the blastomeres will settle down at the bottom by gravity, separating them from yolk globules, which will mainly float on top of the density gradient medium. Transfer blastomeres into 2 mL microcentrifuge tubes. Remove excessive medium, and the cells can be used in various indicated applications. Abbreviations: RNA-seq: RNA sequencing; snRNA-seq: single nuclei RNA sequencing; ATAC-seq: assay for transposase-accessible chromatin using sequencing; RT-PCR: reverse transcription-polymerase chain reaction. Please click here to view a larger version of this figure.

Figure 4: Imaging and measuring the sizes of the sorted single blastomeres. (A) Representative bright-field images of the unsorted (Control) or sorted single blastomeres after isolation from Stage 9 embryos, followed by fixation. The diameters of the single blastomeres (mean ± standard deviation) are labeled on images. Scale bars: 100 µm. (B,C) Measurements of the diameters of the unsorted (Control) and sorted single blastomeres. Data are shown as mean ± standard deviation (B) and violin plots (C) with median (long dashed lines) and quartiles (dashed lines). Numbers of blastomeres analyzed: Control, 489 cells; <40 µm, 1095 cells; 40-70 µm, 562 cells; >70 µm, 241 cells. Please click here to view a larger version of this figure.

Discussion

Cell size is fundamentally important for cellular functions³, and its impact on early development requires more scrutiny in model embryonic systems. Here we have described the details for isolating single cells from Xenopus laevis early embryos and sorting the cells by size using cell strainers, potentially applicable for cell size studies. This method has allowed us to provide the direct evidence of size-regulated zygotic gene activation in early embryogenesis⁴.

Several key steps are critical for the success of the protocol. Because there is a cell-size gradient axis in the Xenopus early embryos - small to large cells from the animal pole to the vegetal pole⁴, the proportion of the cells with various sizes is very different; that is, there are many fewer larger cells than the smaller cells in an embryo. Additionally, during dissociation and sorting, the large cells would continue to divide and tended to burst more frequently than the small cells. Therefore, it is important to estimate the number of embryos needed for blastomere dissociation, and, if specific numbers of cells are required, particularly for large cells, increase the number of embryos to obtain sufficient large blastomeres.

The timing of CMFM incubation depends on the number and stage of embryos and varies under different temperature conditions. Thus, it requires close monitoring during the dissociation process and determining the optimal dissociation time context-dependently, despite an empirical time of 20-30 min⁹. In addition, when transferring the embryos to be dissociated from the 0.1× MMR, which contains both Ca²⁺ and Mg²⁺, to remove their residual amounts, it is recommended to rinse the embryos with some extra CMFM before incubating them with CMFM. Moreover, although regular shaking helps the embryos physically fall apart, it should be done very gently, as the large blastomeres can be easily mechanically damaged or destroyed. Remove any undissociated debris before sorting.

For sorting the blastomeres with cell stainers, here we used the Xenopus embryos at stage 9 as an example and used the cell strainers with the mesh pore sizes of 40 µm, 70 µm, and 100 µm, respectively. We chose those based on our previous discovery of a threshold of cell size at ~40-45 µm that is required for the ZGA onset in vivo, and separating the cell size after the dissociation of blastomeres allowed us to validate the cell size threshold ex vivo⁴. Depending on the experimental needs, one or more of the various-sized cell strainers can be used for embryos at any stages of embryos during early stages of development, ideally from blastula (stage 5) to gastrula (stage 12). As a rule of thumb, the cell strainers with larger mesh pore sizes can be used for the embryos at earlier stages (e.g., 100 µm for stage 5), and the smaller ones can be used for the embryos at earlier stages (e.g., 20 µm for stage 10).

When sorting cells using cell strainers of varying sizes, perform the sorting gently but quickly throughout the process. Because live blastomeres are still dividing during sorting and purification, it is advisable to budget extra time and start earlier than the targeted stage. In addition, during sorting, avoid exposing the blastomeres to the air, which will cause them to lyse. Further, for the blastomeres to settle in the density gradient medium, the speed of sedimentation by gravity depends on their size or weight, and the large blastomeres settle faster than the small ones. Thus, always proceed with additional caution for the large cells.

The size-sorted cells can be used in various applications, including live cell analysis, sequencing and imaging assays. For example, since the individual blastomeres are continually dividing, they can be cultured in vitro, allowing them to be an excellent model for analyzing cell cycles. Also, the nuclei from the live cells can be isolated for sequencing in single-nuclei RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq). Further, the sorted cells can be frozen for RNA work, including reverse transcription polymerase chain reaction (RT-PCR) and bulk RNA-seq. Finally, the sorted cells can be fixed for immunofluorescence and in situ hybridization (ISH). By combining these approaches, the method described here presents an important platform for studying various questions in cellular and developmental biology, particularly those related to cell size regulation.

In summary, this protocol aims to prepare the single cells with varying sizes from early Xenopus embryos. The size-sorted cells can be used for various cellular and molecular studies, such as sequencing and imaging, which could help reveal new mechanisms of development, such as size-dependent gene regulation. This system is also well-suited for studying cell cycle dynamics, constituting an ideal platform for large-scale drug screening and new therapeutic development.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Paraformaldehyde (PFA)	EMS	15710-S	For making the fixative solution
60 mm Disposable Petri Dishes	VWR	25384-092	For incubating embryos
ACCU-SCOPE 3075 Zoom Stereomicroscopes	VWR	470351-130	For visualizing embryos
Agarose	Sigma-Aldrich	A-9414	For coating petri dishes
Cell Strainer, 100 µm	VWR	76327-102	For sorting cells by size
Cell Strainer, 40 µm	VWR	76327-098	For sorting cells by size
Cell Strainer, 70 µm	VWR	76327-100	For sorting cells by size
Disposable Transfer Pipettes	VWR	414004-035	For transferring embryos
Dumont Tweezer, Style 5	Electron Microscopy Sciences	72701-D	For removing vitelline membrane
Glass Petri Dish, 60 × 15 mm	VWR	75845-542	For incubating embryos