Name: Physical Manipulation to Generate Xenopus Mini Embryos (Video) | JoVE Journal
Uploaded: 2026-04-10T13:20:00.000+00:00
Duration: 4 min 9 s
Description: Learn to generate Xenopus laevis mini embryos using hair knot constriction to study cell size effects on zygotic genome activation. This method enables investigation of nucleocytoplasmic ratio regulation in early development.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank the Matthew Good lab at the University of Pennsylvania for providing training. This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03HD105802).

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
<ol>
	<li>Preparing solutions
	<ol>
		<li>Prepare 20&#215; 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.</li>
		<li>Prepare 3% density gradient medium (wt/vol) solution by mixing 1.5 g of density gradient medium into 50 mL of 0.5&#215; MMR. Stir gently with a magnetic stirring bar until the polymer is dissolved. Keep at 4 &#176;C until use.</li>
		<li>Prepare 1% agarose gel in a 60-mm Petri dish by mixing 0.5 g of agarose into 50 mL of 0.1&#215; 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.</li>
	</ol>
	</li>
	<li>Preparing hair knots for constricting embryos
	<ol>
		<li>Get some thin hairs and cut them into ~5 cm pieces.</li>
		<li>Use a tweezer to insert one end of the hair into a P10 tip about ~1 cm deep.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Repeat the above steps to make more hair knots as needed.</li>
	</ol>
	</li>
</ol>
2. Making Xenopus mini embryos
<ol>
	<li>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&#215; MMR, ready for use.</li>
	<li>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 transcripts11,13 before proceeding with the following procedures.</li>
	<li>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&#215; MMR.</li>
	<li>Use a tweezer in one hand to rotate an embryo about ~45&#8211;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat steps 2.4&#8211;2.6 above to make the desired quantity of mini embryos.</li>
	<li>Let the embryos stay in 3% density gradient medium/0.5&#215; MMR for ~1&#8211;2 h. Then, remove the medium with a P1000 pipette without disturbing the embryos and replenish with 15 mL of 0.1&#215; MMR.</li>
	<li>Let the embryos develop into the desired stages, then use tweezers to carefully remove the knots from the mini embryos.</li>
	<li>Collect the embryos for desired analysis, such as visualizing the ZGA in single cells11,13.</li>
</ol>

<ol><li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+major+developmental+transition+in+early+Xenopus+embryos%3A+I.+Characterization+and+timing+of+cellular+changes+at+the+midblastula+stage%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30 (3), 675-686 (1982).</li>
<li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+major+developmental+transition+in+early+Xenopus+embryos%3A+II.+Control+of+the+onset+of+transcription%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription.</a> Cell. 30 (3), 687-696 (1982).</li>
<li>Kane, D. A., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+midblastula+transition%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The zebrafish midblastula transition.</a> Development. 119 (2), 447-456 (1993).</li>
<li>Lu, X., Li, J. M., Elemento, O., Tavazoie, S., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coupling+of+zygotic+transcription+to+mitotic+control+at+the+Drosophila+mid-blastula+transition%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition.</a> Development. 136 (12), 2101-2110 (2009).</li>
<li>Jevtic, P., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Both+nuclear+size+and+DNA+amount+contribute+to+midblastula+transition+timing+in+Xenopus+laevis%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Both nuclear size and DNA amount contribute to midblastula transition timing in Xenopus laevis.</a> Sci Rep. 7 (1), 7908(2017).</li>
<li>Edgar, B. A., Kiehle, C. P., Schubiger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+cycle+control+by+the+nucleo-cytoplasmic+ratio+in+early+Drosophila+development%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cell cycle control by the nucleo-cytoplasmic ratio in early Drosophila development.</a> Cell. 44 (2), 365-372 (1986).</li>
<li>Chen, H., Qian, W., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+cellular+dimensions+with+cell+differentiation+during+early+development%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Integrating cellular dimensions with cell differentiation during early development.</a> Curr Opin Cell Biol. 67, 109-117 (2020).</li>
<li>Lee, D. R., Lee, J. E., Yoon, H. S., Roh, S. I., Kim, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compaction+in+preimplantation+mouse+embryos+is+regulated+by+a+cytoplasmic+regulatory+factor+that+alters+between+1-+and+2-cell+stages+in+a+concentration-dependent+manner%5BTitle%5D)+AND+%22J+Exp+Zool%22%5BJournal%5D)">Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.</a> J Exp Zool. 290 (1), 61-71 (2001).</li>
<li>Ishimatsu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size-reduced+embryos+reveal+a+gradient+scaling-based+mechanism+for+zebrafish+somite+formation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.</a> Development. 145 (11), dev161257(2018).</li>
<li>Clute, P., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+appearance+of+division+asynchrony+and+microtubule-dependent+chromosome+cycles+in+Xenopus+laevis+embryos%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Regulation of the appearance of division asynchrony and microtubule-dependent chromosome cycles in Xenopus laevis embryos.</a> Dev Biol. 171 (2), 273-285 (1995).</li>
<li>Chen, H., Einstein, L. C., Little, S. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+patterning+of+zygotic+genome+activation+in+a+model+vertebrate+embryo%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo.</a> Dev Cell. 49 (6), 852-866.e7 (2019).</li>
<li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+laevis+in+vitro+fertilization+and+natural+mating+methods%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Xenopus laevis in vitro fertilization and natural mating methods.</a> CSH Protoc. 2007, pdb prot4737(2007).</li>
<li>Chen, H., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+nascent+transcription+in+wholemount+vertebrate+embryos+to+characterize+zygotic+genome+activation%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation.</a> Methods Enzymol. 638, 139-165 (2020).</li>
<li>Meaders, J. L., Burgess, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microtubule-based+mechanisms+of+pronuclear+positioning%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Microtubule-based mechanisms of pronuclear positioning.</a> Cells. 9 (2), 505(2020).</li>
</ol>

The author declares no competing interests.

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...

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 motility1,2,3,4. Manipulations of the N:C ratio by changing DNA ploidy support its functional importance in early embryogenesis across several model systems3,4,5,6.
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&#8217;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.

<table><tbody><tr><td>30-G needle</td><td>BD Medical</td><td>305128</td><td>For puncturing embryos</td></tr><tr><td>60 mm Disposable Petri Dishes</td><td>VWR</td><td>25384-092</td><td>For incubating embryos</td></tr><tr><td>ACCU-SCOPE 3075 Zoom Stereomicroscopes</td><td>VWR</td><td>470351-130</td><td>For visualizing embryos</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A-9414</td><td>For coating petri dishes</td></tr><tr><td>Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td>For tweaking and holding embryos</td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>GE17-0300-10</td><td>For incubating embryos</td></tr><tr><td>TipOne Pipette Tip, 10 &amp;micro;L</td><td>USA Scientific</td><td>1111-3700</td><td>For mounting the hair knots</td></tr><tr><td>Wax Block, Black</td><td>Carolina Biological Supply Co.</td><td>974228</td><td>For mounting the hair knots</td></tr></tbody></table>

physical manipulation to generate xenopus mini embryos

Physical Manipulation to Generate Xenopus Mini Embryos

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.

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...

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

A defining characteristic of early embryogenesis is the rapid, exponential decrease in individual cell size while the overall embryo volume remains ...

Our research investigates how cell size regulates early embryonic genome activation by physically generating miniature embryos at the beginning of early development. Existing methods like direct assessment of size impact on genome activation, this method addresses this by directly changing the size of one cell embryo. To begin, prepare hair knots for constricting embryos by cutting thin hair into approximately five centimeter pieces.Using a tweezer, insert one end of the hair into a P10 tip at a depth of about one centimeter. Then take 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 one minute.Ensure the hair is fixed at the end of the P10 tip. Use a tweezer to carefully make an overhand knot of the hair. Adjust the size of the loop to be approximately 1.5 millimeters.Trim the overhanging hair. To begin, obtain Xenopus embryos generated using standard in vitro fertilization procedures and microinjected with five ethanol uridine to label nascent transcripts. Next, add 15 milliliters of 3%density gradient medium containing 0.5x MMR into a previously prepared agarose-covered 60 millimeter Petri dish.At one hour post-fertilization, transfer the embryos into the Petri dish. Under a stereo microscope, use tweezers to rotate the embryo approximately 45 to 60 degrees to access the vegetal pole. Hold the embryo with tweezers and use a 30 gauge needle in the other hand to carefully puncture the vegetal pole.Place the hair knot attached to a P10 tip over the embryo in such a way that the animal pole is enclosed within the hair knot. Adjust the position of the knot according to the size of the mini embryo. Using the P10 pipette tip and tweezers, pull the knot ends in opposite directions to tighten it around the embryo between the animal and vegetal poles.Gently press the embryo and release some cytoplasm. Then cut the hair knot at the point where it attaches to the P10 tip. After generating the required number of mini embryos, let them sit in the 3%density gradient medium for approximately one to two hours.Then using a P1000 pipette, remove the medium without disturbing the embryos and add 15 milliliters of 0.1x MMR. Once the embryos develop into the desired stages, remove the hair knots. The microscopic image of the embryos of obtained at 8.5 hours post-fertilization indicated that the volume of some mini embryos was approximately one-third of that of the control embryos, suggesting that the early Xenopus embryos can be manipulated to generate mini embryos.The most important challenge when performing this protocol is to ligature the one cell embryos to make mini embryos. The mini embryos obtained using this protocol can be used in imaging and sequencing for mechanistic studies. Future studies could explore how embryo and cell size regulate early development and underlying mechanisms.

physical-manipulation-to-generate-xenopus-mini-embryos

Research

JoVE Journal

Developmental Biology

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity.  An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells.  This protocol will help address questions related to spatial control of signaling.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Facial Transplants in Xenopus laevis Embryos

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Quantification of Orofacial Phenotypes in Xenopus

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Functional Cloning Using a Xenopus Oocyte Expression System

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technique to Target Microinjection to the Developing Xenopus Kidney

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Learn to generate Xenopus laevis mini embryos using hair knot constriction to study cell size effects on zygotic genome activation. This method enables investigation of nucleocytoplasmic ratio regulation in early development.

Physical Manipulation to Generate Xenopus Mini Embryos

Explore More Videos