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Front Cell Dev Biol
2025 Dec 18;13:1730288. doi: 10.3389/fcell.2025.1730288.
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Optimized protocols for generating half-sized embryos from separated first two blastomeres in green sea urchin and Xenopus laevis.
Orlov EE
,
Timoshina PS
,
Parshina EA
,
Eroshkin FM
,
Bannikova MA
,
Zaraisky AG
.
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The ability to restore normal body proportions after size reduction is a remarkable feature of early development. At the dawn of experimental embryology, Hans Driesch demonstrated that separated sea urchin blastomeres can develop into fully proportioned organisms, revealing an intrinsic capacity for embryonic scaling. However, the molecular mechanisms that enable this phenomenon remain poorly understood. Modern investigations of scaling, particularly those relying on bulk omics approaches, require reliable methods for producing large numbers of embryos that differ in size from wild-type embryos. Here, we present optimized protocols for generating half-sized embryos from separated blastomeres in two phylogenetically distant model organisms: the green sea urchin (Strongylocentrotus droebachiensis) and the frog (Xenopus laevis). These updated methods build on classical embryological approaches and enable robust, reproducible production of half-sized embryos. The resulting embryos are well suited for downstream applications, including in situ hybridization, morphogen gradient analysis, and high-throughput molecular profiling such as RNA sequencing and proteomics. Together, these protocols offer a powerful platform for investigating the genetic and physical principles that govern embryonic scaling across diverse deuterostome lineages.
FIGURE 1. Generation of Xenopus laevis half-sized embryos. (A) Petri dish with an agarose bed containing rows of wells with wild-type and half-sized embryos. (B) Devitellinized embryo at the first cleavage stage forming a narrow isthmus (arrowheads) between the two blastomeres. (C) Arrow indicates the direction of rotation applied with forceps to stretch the isthmus into a thin, thread-like bridge. (D) Blastomeres immediately after separation. (E) Separated blastomeres placed in agarose wells initiate the second cleavage. Their wild-type sibling in the bottom well is at the same stage. (F) Some half-sized embryos exhibit instability and transient surface damage due to an increased surface-to-volume ratio. (G) Lowering ionic strength promotes re-adhesion of dissociated blastomeres within 10–60 min (H,I) Half-sized embryos at mid-gastrula and early neurula stages, alongside their wild-type siblings. (J) Half-sized and wild-type tadpoles at stage 46. (K,K′)
In situ hybridization using a digoxigenin-labeled probe for the neural plate marker sox2 mRNA reveals perfect scaling of the neural plate in the half-sized embryo at the mid-neurula stage. (L,L′)
In situ hybridization using a digoxigenin-labeled probe for the scaler gene mmp3 mRNA shows a marked decrease in expression in the half-sized embryo compared to its wild-type sibling at the mid-neurula stage. Scale bars: (A) 10 mm; (B–L′) 500 μm.
FIGURE 2. Generation of Strongylocentrotus droebachiensis half-sized embryos. (A) Representative outcome of the blastomere separation procedure at the two-cell stage. Arrows indicate dividing single blastomeres. (B) Wild-type embryo at the mesenchyme blastula stage. (C) Half-sized embryo with an open blastocoel at the early swimming blastula stage. (D) Half-sized embryo with a closed blastocoel at the swimming blastula stage. (E) Half-sized embryo at the mesenchyme blastula stage. (F,G) Full-sized and half-sized pluteus larvae, respectively. (H)
In situ hybridization with a digoxigenin-labeled probe for chordin mRNA reveals correct scaling of the chordin expression domain in half-sized embryos at the mesenchyme blastula stage. (I)
In situ hybridization with a digoxigenin-labeled probe for bp10 mRNA shows enhanced expression of this scaler gene in half-sized embryos at the mesenchyme blastula stage. In situ hybridization were performed as described in Timoshina et al. (2024). Scale bars: (A) 60 μm; (D,E,G) 25 μm; (F) 30 μm; (H,I) 120 μm.
FIGURE 3. Results of separation and cultivation of Xenopus laevis blastomeres according to the protocols described in Cooke and Webber (1985a), Cooke and Webber (1985b), and Kageura and Yamana, 1983. (A) Results of blastomere separation in eight 2-cell stage embryos (A1–A8) as described in Cooke and Webber (1985a) and Cooke and Webber (1985b), namely by gently blowing the solution from a fine pipette onto the new membrane in the deepening furrows. In all cases, this resulted in either rupturing of the blastomeres or the blastomeres remaining attached to each other if the flow of medium from the pipette was weak. Embryos are shown at 4 cells stage. (B) Eight blastomeres obtained by separating embryos at the 2-cell stage were cultured as described by Kageura and Yamana (1983) namely in wells of a tissue culture plate (Corning analog of Falcon-3034) with the bottom covered with 2% agar. Beginning at the two-cell stage, blastomeres gradually lost contact with each other. As a result, by the sixteen-cell stage, embryos 1–8 shown in (B) consisted of loosely connected cells and had a flattened shape. Soon after, the blastomeres began to die. Note that the cells appear slightly pink because, following the protocol described by Kageura and Yamana (1983), they were temporarily cultured in 50% Leibovitz (L-15) medium containing phenol red dye.
