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 support its functional importance in early embryogenesis across several model systems^3,4,5,6.

Physical manipulation of embryo size represents an important technique for studying regulatory mechanisms in early embryonic development⁷. Cytoplasmic removal by aspiration, which alters the N:C ratio in early mouse embryos, induces premature compaction⁸. Moreover, reducing zebrafish embryo size by chopping revealed a new gradient-based mechanism of somite scaling⁹. Furthermore, reducing cytoplasmic volume in Xenopus embryos revealed N:C ratio-dependent cell cycle dynamics¹⁰. Importantly, by generating Xenopus mini embryos through cell size reduction and labeling nascent transcripts with 5-ethynyl-uridine (5-EU) in single cells, we demonstrated that reducing cell size induces zygotic genome activation (ZGA) in a dose-dependent manner¹¹. These results support the crucial role of cell size in early embryogenesis.

Here, we describe a detailed procedure for generating Xenopus laevis mini embryos by physical manipulation using hair knots (Figure 1)^10,11. This approach builds on Newport and Kirschner’s (1982) foundational use of hair loop constriction on early Xenopus embryos¹, providing a clear, reliable protocol for generating mini embryos as experimental tools. Additionally, this method is applicable to other embryonic systems⁷, which would facilitate uncovering new mechanisms of early embryogenesis.

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 °C until use.
   3. Prepare 1% agarose gel in a 60-mm Petri dish by mixing 0.5 g of agarose into 50 mL of 0.1× MMR. Heat with a microwave oven to dissolve agarose, then add 5 mL of agarose to coat the bottom of a 60-mm Petri dish in a thin layer.
2. Preparing hair knots for constricting embryos
   1. Get some thin hairs and cut them into ~5 cm pieces.
   2. Use a tweezer to insert one end of the hair into a P10 tip about ~1 cm deep.
   3. Put wax on a spoon and heat it to melt the wax. Quickly dip the hair-attached P10 tip in the wax, and let it dry for 1 min. Ensure the hair is fixed at the end of the P10 tip.
   4. Use a tweezer to carefully make an overhand knot of the hair. Adjust the size of the loop to be ~1.5 mm. Trim the overhang if needed. Put the knot aside for future use.
   5. Repeat the above steps to make more hair knots as needed.

2. Making Xenopus mini embryos

1. To obtain Xenopus embryos, follow a standard protocol to perform in vitro fertilization (IVF) (for example, see Sive et al., 2007)¹². The embryo should be in 0.1× MMR, ready for use.
2. To visualize ZGA in single cells of early embryos and study their regulatory mechanisms, microinject the 1-cell stage embryos with 5-ethynyl-uridine (5-EU) to metabolically label the nascent transcripts^11,13 before proceeding with the following procedures.
3. When embryos are at ~1 hour post-fertilization (hpf), use a plastic transfer pipette to transfer ~20 embryos in a 60-mm Petri dish (covered with 1% agarose gel) containing ~15 mL of 3% density gradient medium/0.5× MMR.
4. Use a tweezer in one hand to rotate an embryo about ~45–60 degrees under a stereomicroscope to access the vegetal pole (the pale hemisphere). Hold the tilted embryo with the tweezers and use a 30-G needle in the other hand to carefully puncture the vegetal pole. Release some cytoplasm by gently pressing the embryo.
5. Carefully cover a hair knot mounted in a P10 tip on an embryo so that the animal pole (the dark hemisphere) will be inside the hair knot. Adjust the position of the knot according to the desired mini embryo size.
6. Hold the P10 pipette with one hand and use a tweezer on the other hand to pull the two overhand ends of the knot in opposite directions. The hair knot will tighten around the embryo, between the animal pole (intact) and the vegetal pole (with cytoplasm released). Cut off the hair knot at the site where it attaches to the P10 tip.
7. Repeat steps 2.4–2.6 above to make the desired quantity of mini embryos.
8. Let the embryos stay in 3% density gradient medium/0.5× MMR for ~1–2 h. Then, remove the medium with a P1000 pipette without disturbing the embryos and replenish with 15 mL of 0.1× MMR.
9. Let the embryos develop into the desired stages, then use tweezers to carefully remove the knots from the mini embryos.
10. Collect the embryos for desired analysis, such as visualizing the ZGA in single cells^11,13.

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