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....

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 KH₂PO

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 i.......

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.

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