Dorsal Axial Structures Research Articles

The Location of Dorsal Information in Frog Early Development: (dorsoventral polarity/organizer/mesoderm/dorsal).

Dorsal information is necessary for the development of the dorsal axial structures which characterize the vertebrates. The nature of dorsal information in early embryos is not known, but its presence is required for the formation of dorsal mesoderm with Spermann organizer activity. In frogs, the dorsoventral axis is specified by a cortical/cytoplasmic rotation in the egg shortly after fertilization, and this dorsal information is limited to a few cells in the equatorial and vegetal region of early cleavage embryos. At the 8-cell stage, 2-4 cells can promote dorsal development, and at the 32-cell stage, 4-6 cells have dorsal information. Recent experiments have shown that growth factors can induce cells to form dorsal mesoderm and that lithium can act in those cells to enhance the induction. It will be important to determine the relationship between the location of dorsal information defined embryologically and factors involved in the development of dorsal mesoderm.

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis: (regulation/Xenopus laevis/cell-deficient embryo/dorsal axial structures).

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis. Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells. These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.

The correlation between patterns of dye transfer through gap junctions and future developmental fate in Xenopus: the consequences of u.v. irradiation and lithium treatment

ABSTRACT The correlation between cell-to-cell communication through gap junctions at the 32-cell stage and the subsequent patterning of the embryonic axis has been examined in Xenopus laevis embryos. Disturbances of embryonic axis formation were generated by exposure to u.v. irradiation at the vegetal pole before 0·6 in the cell cycle, which generates embryos with dorsal axial deficiencies. Alternatively embryos were treated with 100 mm-lithium chloride between the 2-cell and 32-cell stage, which generates embryos with ventral axial deficiencies. The cell-to-cell transfer of Lucifer Yellow was used to monitor junctional permeability. Injections were made into animal hemisphere cells, lying in tiers 1 and 2 of the 32-cell embryo, whose position relative to the future dorsoventral axis of the embryo was determined on the basis of differences in pigmentation. The frequency of Lucifer Yellow transfer in the future dorsal half of the animal hemisphere was compared with that in the future ventral half for control (untreated), u.v.-irradiated and Li-treated embryos. Injected embryos were subsequently scored for axial development for comparison with dye transfer frequencies. In control embryos at the 32-cell stage, Lucifer Yellow transfer was both more frequent and more extensive in future dorsal regions than in future ventral regions, as observed previously. In embryos that had been u.v. irradiated before 0·6 in the first cell cycle, Lucifer transfer was the same in both light and dark regions of the animal hemisphere and at the low level characteristic of future ventral regions in normal embryos. These embryos developed with massive reductions in dorsal axial structures. Embryos irradiated after 0-8 in the first cell cycle, when u.v. irradiation no longer inhibits the cytoplasmic movements initiated at fertilization, showed a normal dorsoventral difference in Lucifer Yellow transfer and developed with normal dorsoventral polarity. Embryos exposed to 100mm-LiCl contained 1·4 mm-lithium at the end of the treatment period. The transfer of Lucifer Yellow between animal pole cells of 32-cell embryos previously treated with LiCl was the same in both light (dorsal) and dark (ventral) and at the high level characteristic of future dorsal regions in normal embryos. Such embryos subsequently developed with substantial ventral axis deficiencies. U.v.-irradiated embryos subsequently treated with lithium showed transfer rates in ventral regions equivalent to that in embryos treated with Li alone and developed with ventral axial deficiencies. We conclude that the frequency with which Lucifer Yellow transfers through gap junctions at the 32-cell stage indicates the future developmental fate of animal cells and can be used to predict the subsequent organization of the embryonic axis.

Dorsalization of mesoderm induction by lithium

Lithium dorsalizes the body plan of Xenopus embryos when administered at the 32-cell stage (K. R. Kao and R. P. Elinson, 1988, Dev. Biol. 127, 64–77) . In this paper, we have attempted to determine the effects of lithium on mesoderm induction, in order to localize the target of action of lithium. In the 32-cell embryo, the vegetal-most tier 4 cells are able to induce dorsal development in the overlying, equatorial tier 3 cells (R. L. Gimlich and J. C. Gerhart, 1984, Dev. Biol. 104, 117–130) . Our experiments show that microinjection of lithium into either tier 3 or tier 4 cells of ultraviolet-irradiated, dorsoanterior-deficient embryos rescues normal development. Lineage tracer studies show that only tier 3-injected cells contribute progeny to dorsal axial structures while tier 4-injected cells contribute progeny to endoderm. Sandwich explants between animal caps and ventral vegetal cells cause induction of large amounts of muscle in the explants if either animal caps or vegetal cells are pretreated with lithium. Similarly, fibroblast growth factor-mediated mesoderm induction is also modified by lithium so that muscle is induced instead of ventral mesoderm. We conclude that lithium dorsalizes the response of animal cells to mesoderm induction signals, while not acting directly as a mesoderm inducer itself. The target of action of lithium is likely the third tier of cells of the 32-cell embryo.

The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland ( K. R. Kao, Y. Masui, and R. P. Elinson, 1986, Nature (London) 322, 371–373 ). In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.

Fate map for the 32-cell stage of Xenopus laevis.

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells: (cell lineage/chimera/hybrid/Xenopus/dorsalization).

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis. The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.

Pattern formation in 8-cell composite embryos of Xenopus laevis

We have shown in defect experiments that an 8-cell embryo of Xenopus laevis consists of three kinds of cells, that is, animal, vegetal dorsal and vegetal ventral cells, and that cells of different kinds are distinctly different in their developmental capacity. Complete pattern formation occurs in any defect embryo which contains at least two animal, one vegetal dorsal and one vegetal ventral cell. In the present transplantation experiments, we replaced one or two cells of one 8-cell embryo by those of another to obtain 29 series of composite embryos, in which the cell composition of an embryo and/or the dorsoventral orientation of individual cells differed from those of a normal 8-cell embryo. The resulting embryos were examined macroscopically when controls reached stage 26 (tailbud stage) and later. The results showed that both the two animal dorsal cells or one vegetal dorsal cell could be replaced by animal ventral cells or a vegetal ventral cell, respectively, without any detectable effect on pattern formation, irrespective of the ventrodorsal direction of the ventral cells. On the other hand, replacement of an animal ventral or a vegetal ventral cell by an animal dorsal or a vegetal dorsal cell, respectively, made most composite embryos twins. Twins were also formed when a left-handed vegetal dorsal cell was replaced by a right-handed counterpart and vice versa. In these composite embryos, the dorsoventral orientation of the transplanted cell was different from that of a resident dorsal cell or cells of a recipient, and several lines of evidence show that the dorsal cell transplanted in an off-axis orientation is responsible for twin formation. Thus, dorsal cells have the capacity to form dorsal axial structures at later stages and this capacity is localized on the dorsal side, and endows the cells with polarity. On the other hand, ventral cells did not have this capacity or polarity, judging from the fact that their orientation had no effect on pattern formation. One vegetal dorsal or ventral cell could be replaced by an animal dorsal or ventral cell, respectively, without any marked effect. However, replacement of two vegetal cells by animal ones and of one or two animal cells by vegetal ones resulted in deficiency of vegetal cells and oedema and in deficiency of animal cells and incomplete invagination, respectively. Twin formation in composite embryos with animal dorsal cells in place of animal ventral ones is discussed in consideration of findings in recombination experiments by Nieuwkoop.

Further analysis of the effect of ultra-violet irradiation on the formation of the germ line inXenopus laevis

Ultra-violet (u.v.) irradiation of the vegetal pole of newly fertilized eggs has three documented effects: reduction of primordial germ cells (PGCs), cytological damage to the vegetal hemisphere and disruption of the normal mechanism by which the vegetal yolk mass induces the formation of the dorsal axis of the embryo. In this study, we find that 90 degrees rotation of the egg for various periods after irradiation rescues the dorsal axial structures but does not restore the number of PGCs found in the dorsal mesentery of the gut; neither is there any correlation between reduced numbers of PGCs and disruption of cleavage at the vegetal pole. We therefore conclude that the effect on the germ line is separate from the other two phenomena. Secondly, 90 degrees rotation of non-irradiated eggs was found to significantly reduce germ cell numbers migrating in the dorsal mesentery of the gut.