High-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage

doi:10.1016/0012-1606(86)90059-X

Developmental Biology

Volume 116, Issue 1, July 1986, Pages 228-240

https://doi.org/10.1016/0012-1606(86)90059-X Get rights and content

Abstract

In embryos of the frog Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity and centrifugal force, applied at an angle of 90° to the animal-vegetal axis of the egg, can override this topographic relationship and can cause the dorsal structures to be positioned according to the force vector (S. Black and J. Gerhart, 1985, Dev. Biol. 108, 310–324). We report here that at time 0.40 (40% of the first cleavage interval), an average of 60% of eggs centrifuged at 30g for 4 min in this orientation form conjoined twins with one body axis arising from the centripetal side of the egg and one arising from the centrifugal side of the egg. This positioning is observed regardless of the orientation of the side of sperm entry in the centrifugal field. If, after the 0.40 centrifugation, the eggs are inclined with the centripetal side up, they do not make twins; instead, they make only a single axis at the centripetal side. This indicates that the second axis in twins is caused to form by postcentrifugation gravity-driven internal rearrangements of materials that were displaced by the centrifugation. Twins also form at high frequency in eggs centrifuged twice, first at an inclination of 90°, and then at an inclination of 0°. The second centrifugation yields secondary axes even when it is begun midway in the second cell cycle, well after the time of grey crescent formation. Double centrifugation also causes twinning (“double rescue”) of uv-irradiated eggs which otherwise would not develop axial structures. This suggests that the internal displacements caused by the centrifugations can substitute for a step in the normal axis specification process that is impaired in irradiated eggs.

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The number of linearly independent vector fields on a sphere is finite (Adams, 1962). Some of these topologies can be expected to be compatible with twins (Black and Gerhart, 1986) or even multiple embryos forming from one egg (as in armadillos: Section 1.03 in Gordon, 1999), and others might thereby correspond to arrested development. ( cf. Boryskina et al., 2011; Fleury and Gordon, 2012).
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This process is best understood in Xenopus. In frogs, determinants of the major axes are localized to the vegetal pole and are activated after fertilization (Black and Gerhart, 1986; Scharf and Gerhart, 1983). Rotation of the cortical cytoplasm during the first cell cycle transports components of the Wnt pathway, such as Disheveled and β-catenin, from the vegetal pole to the dorsal blastomeres (Miller et al., 1999; Rowning et al., 1997).
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Effects of hypergravity environments on amphibian development, gene expression and apoptosis
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This study investigates how rearing under conditions of hypergravity affects amphibian development, Xotx2 and Xag1 gene expression and apoptosis. Uncleaved Xenopus laevis eggs 20 min after insemination, 2 cell stage embryos, and gastrula stage embryos were raised at 2G and 5G, while controls were raised in normal gravity. Apoptosis in brain and eye inner structures of hatching embryos was scored using the TUNEL staining method, and gene expression in tail-bud embryos was analyzed by whole-mount in situ hybridization. Results showed that: (1) 5G retarded the development of eggs and embryos and induced microcephaly and microphthalmia. (2) 5G suppressed the expression of the two genes, Xotx2 (involved in fore- and midbrain and eye development) and Xag1 (regulating cement gland formation). (3) Eggs and 2 cell stage embryos raised at 5G showed a greater extent of brain and eye apoptosis compared with controls, while those raised at 2G showed no significant difference. These findings suggest that high gravity suppresses certain gene functions and induces abnormal apoptosis in brain and eyes, resulting in developmental retardation and various morphological abnormalities.
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Amphibian embryos have served as a model system for vertebrate axial patterning for more than a century. Recent changes to the Xenopus laevis fate map revised the assignment of the embryonic dorsal/ventral (back-to-belly) axis in pre-gastrula embryos and allowed the assignment of the rostral/caudal (head-to-tail) axis for the first time. Revising the embryonic axes after many years of experimentation changes our view of axial patterning in amphibians. In this review, we discuss the revised maps and axes, and show by example how the new map alters the interpretation of three experiments that form the foundations of amphibian embryology. We compare the revised amphibian fate map to the general maps of the protochordates, and discuss which features of the maps and early development are shared by chordates and which distinguish vertebrates. Finally, we offer an explanation for the formation of both complete and incomplete axes in the rescue assays routinely used to study axial patterning in Xenopus, and a model of amphibian axial patterning.
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However, it is unclear how the forces required to artificially deform the embryo lead to embryonic epithelium stresses and strains that are related to endogenous forces and deformations present in the embryo during development. Importantly, the mechanical induction of Twist is independent of the maternal determinants of dorsal-ventral polarity [29]. Instead, I show that this induction depends on the nuclear translocation of Armadillo and its ability to activate transcription.
Background: Morphogenetic movements are closely regulated by the expression of developmental genes. Here I examine whether developmental gene expression can in turn be mechanically regulated by morphogenetic movements. I have analyzed the effects of mechanical stress on the expression of Twist, which is normally expressed only in the most ventral cells of the cellular blastoderm embryo under the control of the Dorsal morphogen gradient. At embryogenesis gastrulation (stage 7), Twist is also expressed in the anterior foregut and stomodeal primordia.
Results: Submitting the early Drosophila embryo to a transient 10% uniaxial lateral deformation induces the ectopic expression of Twist around the entire dorsal-ventral axis and results in the ventralization of the embryo. This induction is independent of the Dorsal gradient and is triggered by mechanically induced Armadillo nuclear translocation. I also show that Twist is not expressed in the anterior foregut and stomodeal primordia at stage 7 in mutants that block the morphogenetic movement of germ-band extension. Because I can rescue the mutants with gentle compression of these cells, my interpretation is that the stomodeal-cell compression normally caused by the germ-band extension induces the expression of Twist. Correspondingly, laser ablation of dorsal cells in wild-type embryos relaxes stomodeal cell compression and reduces Twist expression in the stomodeal primordium. I also demonstrate that the induction of Twist in these cells depends on the nuclear translocation of Armadillo.
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^☆: This research was supported by USPHS Grant GM 19363, NASA Research Interchange NCA2-1R050404, and NIH Traineeship GM 07127.

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Full paperHigh-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage☆

Abstract

Dev. Biol

Dev. Biol

Dev. Biol

Dev. Biol

Dev. Biol

Dev. Biol

Dev. Biol

Adv. Morphog

Dev. Biol

Dev. Biol

Dev. Biol

Exp. Cell Res

Recherches sur le déterminisme de la symétrie bilatérale dans l'oeuf de Amphibiens

Bull. Biol. Fr. Belg. Suppl

Über den Einfluss der Schwere auf das Froschei

Arch. Mikrosk. Anat

Cortical grafting in Xenopus laevis

J. Embryol. Exp. Morphol

Morphogenetic interactions before gastrulation in the amphibian, Xenopus laevis—The cortical field

J. Embryol. Exp. Morphol

Mechanisms regulating pattern formation in the amphibian egg and early embryo

A reinvestigation of the role of the grey crescent in axis formation in Xenopus laevis

Nature (London)

Cellular and pancellular organization of the amphibian embryo

Full paper
High-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage☆