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Einsteck procedure refers to a method whereby the experimenter inserts material into the blastocoel cavity of an early amphibian embryo. This procedure is simpler to perform than other types of grafts, such as Spemann-Mangold, and with practice yields a sizable amount of data suitable for statistical analysis. This protocol for Einsteck transplantation in Xenopus describes the insertion of the gastrula-stage blastopore lip into the blastocoel cavity of a host embryo., (© 2019 Cold Spring Harbor Laboratory Press.)

In 1924, Hans Spemann and Hilde Mangold (née Pröscholdt) published their famous work describing the transplantation of dorsal blastopore lip of one newt gastrula embryo onto the ventral side of a host embryo at the same stage. They performed these grafts using two newt species with different pigmentation ( Triturus taeniatus and Triturus cristatus ) to follow the fate of the grafted tissue. These experiments resulted in the development of conjoined twins attached through their belly. Because of the difference in embryo pigmentation between the two Triturus species, they determined that the bulk of the secondary embryo arose from the host embryo while the grafted tissue per se gave increase to the notochord and a few somitic cells. This meant that the dorsal blastopore lip was able to organize an almost complete embryo out of ventral tissue. The dorsal blastopore lip is now called the Spemann-Mangold organizer. Here, we describe a simple yet efficient protocol to perform these grafts using the anuran Xenopus laevis., (© 2019 Cold Spring Harbor Laboratory Press.)

The fates of individual cleavage-stage blastomeres and of groups of cells at the blastula through gastrula stages of Xenopus embryos have been mapped in great detail. These studies identified the major contributors of the three germ layers as well as a variety of tissues and organs and several specific cell types. One can use these fate maps to test the commitment of single cells or groups of cells to produce their normal repertoire of descendants, to identify the genes that regulate fate commitment, and to modulate the levels of gene expression in specific lineages to determine gene function in a variety of developmental processes. Here we introduce methods that include how to identify specific blastomeres, inject them with lineage tracers, and alter gene expression levels. We also discuss methods for assaying protein and mRNA expression in situ and for providing novel embryonic environments to test fate commitment. These techniques draw upon classical approaches that are quite easy to perform in the versatile Xenopus embryo., (© 2019 Cold Spring Harbor Laboratory Press.)

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis , depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 μN during gastrulation and over 4 μN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 μN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms., Competing Interests: DS, EK, LD, RK No competing interests declared, (© 2018, Shook et al.)

We have previously shown that the cell sorting process of animal pole cells (AC) and vegetal pole cells (VC) from Xenopus gastrulae is considered to involve two steps: concentrification and polarization. In this study, we addressed the question of what specified the spatial relationship of the AC and VC clusters during the process. First, we examined the inhibitory or facilitatory treatment for myosin 2 activity during each of the two steps. The aggregates treated with Y27632 or blebbistatin during the concentrification step showed a cluster random arrangement, suggesting the prevention of the cell sorting by inhibition of myosin 2. Meanwhile, the treatment with a Rac1 inhibitor, NSC23766, during the same step resulted in promotion of the fusion of the AC clusters and the progression of the cell sorting, presumably by an indirect activation of myosin 2. On the other hand, the treatments with any of the three drugs during the polarization step showed that the two clusters did not appose, and their array remained concentric. Thus, the modulation of cell contraction might be indispensable to each of the two steps. Next, the activin/nodal TGF-β signaling was perturbed by using a specific activin receptor-like kinase inhibitor, SB431542. The results revealed a bimodal participation of the activin/nodal TGF-β signaling, i.e., suppressive and promotive effects on the concentrification and the polarization, respectively. Thus, the present in vitro system, which permits not only the cell contraction-mediated cell sorting but also the TGF-β-directed mesodermal induction such as cartilage formation, may fairly reflect the embryogenesis in vivo.