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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.
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Figure 1: Example of dissociated blastomeres. Image shows blastomeres dissociated at stage 9. Note the variation in the blastomere size. Scale bar: 100 µm.
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.
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.
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.
