Physical Manipulation to Generate Xenopus Mini Embryos

Summary

We describe a physical constriction method to make mini embryos from Xenopus laevis 1-cell embryos.

Abstract

A defining characteristic of early embryogenesis is the rapid, exponential decrease in individual cell size while the overall embryo volume remains constant. The nucleocytoplasmic (N:C) ratio plays a critical role in regulating cellular and molecular activities essential to early embryogenesis. Physical manipulation of the embryo size, therefore, represents an important way of investigating these developmental mechanisms. The large size (~ 1.2 mm in diameter) and high developmental plasticity of Xenopus laevis early embryos make them an ideal model for physical dissection and manipulation to interrogate the regulatory mechanisms of development. Here, we describe an approach for generating mini embryos with altered cytoplasmic volume and N:C ratio by constricting 1-cell Xenopus laevis embryos with hair knots. The resulting mini embryos have been shown to initiate early zygotic genome activation (ZGA) directly induced by cell size reduction and N:C ratio growth. These mini embryos, which can be generalized to other embryonic systems, therefore provide a unique, powerful tool for dissecting size-mediated mechanisms of early embryo development.

Introduction

One of the most striking features of early embryogenesis is the exponential reduction of individual cell size within an embryo that maintains a constant volume. Rapid DNA synthesis and cell size reduction lead to an increase in the nucleocytoplasmic ratio (N:C ratio), which plays an important role in regulating several hallmark activities of the mid-blastula transition (MBT) in model embryonic systems, including zygotic gene transcription, cell cycle elongation, and cell motility^1,2,3,4. Manipulations of the N:C ratio by changing DNA ploidy ....

Protocol

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

1. Preparing materials

1. Preparing solutions
   1. Prepare 20× MMR by mixing 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 3% density gradient medium (wt/vol) solution by mixing 1.5 g of density gradient medium into 50 mL of 0.5× MMR. Stir gently with a magnetic stirring bar until the polymer is dissolved. Keep at 4 °....

Results

Xenopus laevis embryos at the 1-cell stage were constricted with the hair knots as described above. The control and mini embryos at ~Stage 9 (8.5 hpf at 23 °C) were imaged under a stereomicroscope. As shown in Figure 2, the volume of some mini embryos is ~1/3 of that of the control embryos. This result suggests that the early Xenopus embryos can be manipulated to generate mini embryos, providing a unique model for studying the impact of cell size on the dynamics of cell cyc.......

Discussion

Here, we describe a method for generating mini-embryos in Xenopus by physically constricting 1-cell stage embryos with hair knots. This approach provides direct evidence that reducing cell size induces ZGA in a model embryo¹¹.

Several aspects of this protocol are critical to successfully generate mini-embryos. To make hair knots, it is important to use thin, durable hair, preferably that of babies. The thicker and more fragile the hairs are, the harder it is to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30-G needle	BD Medical	305128	For puncturing embryos
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
Dumont Tweezer, Style 5	Electron Microscopy Sciences	72701-D	For tweaking and holding embryos
Ficoll 400	Sigma-Aldrich	GE17-0300-10	For incubating embryos
TipOne Pipette Tip, 10 µL	USA Scientific	1111-3700	For mounting the hair knots
Wax Block, Black	Carolina Biological Supply Co.	974228	For mounting the hair knots