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Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.
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???displayArticle.pmcLink???PMC7032064 ???displayArticle.link???Int J Dev Biol ???displayArticle.grants???[+]
Fig. 1. Diagram of the experimental procedure used to investigate regeneration in Xenopus laevis half embryos. On the left, drawing of blastula
embryos being bisected in the sagittal or Dorsal/Ventral directions. Middle, the five samples used to generate RNA-seq libraries. (A) Uncut whole
embryo. (B) Right half embryo cut sagittally; note that the maternal pigment is darker on the left side of the tailbud tadpole (while it is uniform in the
whole embryo). (C) Left half from the same blastula; note that pigment asymmetry is concentrated on the right side. (D) Dorsal half embryo at tailbud
showing almost perfect scaling of the half embryo along the antero-posterior axis. (E) Ventral half embryo; because it lacks the Spemann organizer
it develops into a belly-piece consisting of ventral tissues, such as blood and lateral plate, without any dorsal axis (Spemann, 1938). Photographs for
right and left half tailbud embryos are reproduced, with permission, from Moriyama and De Robertis, 2018, while dorsal and ventral half embryos are
unpublished but from the same experimental batch. On the right-hand side, diagrams showing how the displacement by 90° of the Spemann organizer
explains the pigment asymmetry observed in twinned embryos (Moriyama and De Robertis, 2018). Numbers indicate the four segments into which the
16-cell embryo can be lineage-traced. After bisection the dorsal-most segment 1 becomes juxtaposed (at the wound healing point) to the ventral-most
segment 4, which has high BMP and Wnt expression potential. The organizer is not yet formed at midblastula, but at early gastrula the new dorsal lip
(organizer indicated by the red dot) forms 90° away from its original D-V location. Embryos in which both halves heal within 60 minutes and in which the
organizer forms 90° away from the healing point have a high percentage (around 80%) of correct twinning. Segments 4 and 3 form the epidermis, and
involution (white arrow) of the displaced organizer splits them into a less pigmented and more pigmented half, thus explaining the intriguing pigmenta-
tion asymmetry observed (Moriyama and De Robertis, 2018). Note that in the dorsal half embryo pigmentation is symmetric. Since individual embryos
from this five conditions were sequenced (107 100 bp reads) in triplicate at early and late gastrula, the data for 16,000 gene transcripts presented in the
searchable Excel Tables S1 and S2 represents a treasure trove of gene expression at the gastrula stage.
Fig. 2 Heatmap of genes up-regulated by sagittal bisection in regenerating stage 12 late gastrula
Xenopus laevis embryos. Heatmap showing the horizontal Row Z-scores of genes bisected at stage
8 and cultured until stage 12. Standard deviations were calculated from RPKMs of transcripts in left,
right, and dorsal/ventral halves (see also Table 2). Highly expressed genes are shown in blue and lower
ones in pink
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