Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo

This paper describes a continuing effort to define the location and mode of action of morphogenetic determinants which direct the development of dorsal body axis structures in embryos of the frog Xenopus laevis. Earlier results demonstrated that presumptive endodermal cells in one vegetal quadrant of the 64-cell embryo can, under certain experimental conditions, induce partial or complete body axis formation by progeny of adjacent equatorial cells. (R.L. Gimlich and J.C. Gerhart, 1984, Dev. Biol. 104, 117-130). I have now assessed the importance of other blastomeres for embryonic axis formation in a series of transplantation experiments using cells from the equatorial level of the 32-cell embryo. The transplant recipients were embryos which had been irradiated with ultraviolet light before first cleavage. Without transplantation, embryos failed to develop the dorsal structures of the embryonic body axis. However, cells of these recipients were competent to respond to inductive signals from transplanted tissue and to participate in normal embryogenesis. Dorsal equatorial cells, but not their lateral or ventral counterparts, often caused partial or complete body axis development in irradiated recipients, and themselves formed much of the notochord and some prechordal and somitic mesoderm. These are the same structures that they would have formed in the normal donor. Thus, the dorsal equatorial blastomeres were often at least partially autonomous in developing according to their prospective fates. In addition, they induced progeny of neighboring host cells to contribute to the axial mesoderm and to form most of the central nervous system. The frequency with which such transplants caused complete axis formation in irradiated hosts increased when they were made at later and later cleavage stages. In contrast, the inductive activity of vegetal cells remained the same or declined during the cleavage period. These and other results suggest that the egg cytoplasmic region containing "axial determinants" is distributed to both endodermal and mesodermal precursors in the dorsal-most quadrant of the early blastula.     

Early cellular interactions promote embryonic axis formation in Xenopus laevis

We have attempted to define the location and mode of action of axial determinants in the egg of Xenopus laevis. To this end, we transplanted small numbers of blastomeres from normal 64-cell stage embryos into synchronous recipient embryos which had been irradiated with ultraviolet light prior to first cleavage. Without transplantation, such embryos fail to develop dorsal structures of the embryonic body axis. We found that one to three blastomeres transplanted from the vegetal-most octet of cells can effect complete or partial rescue of of axis development in a recipient, provided that the donor cells derive from the quadrant just under the prospective dorsal marginal region. These same cells, when transplanted into the ventral vegetal quadrant of a normal 64-cell embryo, cause the formation of a complete second body axis. In contrast, other cells from the vegetal octet of normal donors fail to cause axis formation. When the rescuing donor cells are labeled with a lineage-restricted fluorescent marker, we find that their progeny do not contribute to the axial structures of the recipient. Progeny of the transplanted cells are found below the level of the blastopore in the early gastrula and eventually give rise to portions of the gut, as is their fate in normal development. These results, in agreement with those of Nieuwkoop (P.D. Nieuwkoop, 1977, Curr. Top. Dev. Biol. 11, 115-132), imply that the dorsal-most vegetal cells of the 64-cell embryo receive from the egg cytoplasm a set of determinants enabling them to induce neighboring cells to undertake axis formation. We discuss the relationship between axis induction in rescued irradiated embryos and axis determining processes in normal embryogenesis.     

Dorsal Blastomeres in the Equatorial Region of the 32-Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32-cell Xenopus embryos, their roles in head formation in normal development and the organizer-inducing capabilities of the dorsal-most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis . When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32-cell stage and comes into existence not under the inductive influence of the dorsal-most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32-cell embryo.     

Cytoplasmic and molecular reconstruction of Xenopus embryos: synergy of dorsalizing and endo-mesodermalizing determinants drives early axial patterning

Ablation of vegetal cytoplasm from newly fertilized Xenopus eggs results in the development of permanent blastula-type embryos (PBEs). PBEs cleave normally and develop into a very simple tissue consisting only of atypical epidermis. We tried to restore complete embryonic development in PBEs by cytoplasmic transplantation or by mRNA injection. We show a two-step reconstruction of the body plan. In the first step, PBEs injected with either marginal cytoplasm or synthetic VegT RNA restored gastrulation and mesoderm formation, but not axial patterning. Injection of Xwnt8 mRNA (acting upstream of beta-catenin and thus substitutes for the dorsal determinant) did not restore axial development in PBEs. Simultaneous injections of Xwnt8 and VegT into PBEs resulted in dorsal axis development, showing the synergy of these molecules in axial development. These results suggest that the mixing of two cytoplasmic determinants, i.e. the dorsal determinant in the vegetal pole and the endo-mesodermal determinant in the whole vegetal half, triggers the early axial developmental process in Xenopus embryos.     

16细胞期爪蟾胚胎背方与腹方裂球的发育能力

在两栖类爪蟾胚胎发育中,由受精引起的皮层转动造成了受精卵的背腹极性。为了研究受精卵细胞质的不均一分布对胚胎体轴形成的影响,我们进行了16细胞期动物极背、腹方裂球的外植和异位移植实验。16细胞期的动物极背方裂球在外植和移植到腹方位置后都表现出背方特征,如外植块培养到原肠中期时伸长,背方裂球在移植到腹方后引发第二体轴等;而16细胞期动物极腹方裂球移植到背方后其发育命运则遵循背方裂球的命运,参与背方结构的形成。我们认为在16细胞期,动物极背、腹方的裂球由于包含着不同的卵质,因而在发育能力上已经具有背、腹的差异。     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originatefrom the axis of organelles and cytoskeletal elements establishedin the oocyte as it divides from the oogonium. Along this axis,cytoplasmic materials are localized during oogenesis: yolk platelets,for example, are translocated toward the vegetal pole, increasingtheir amount and size in that region. In the first cell cycleafter fertilization, the egg cortex rotates 30° relativeto the cytoplasmic core, modifying animal-vegetal organization.The direction of this rotation, biased by the point of spermentry, defines the site of development of anatomical structuresof the dorsal midline of the embryo. As its immediate effect,rotation activates the cytoplasm of a subregion of the vegetalhemisphere, causing cells cleaved from this subregion to bemore effective than other vegetal parts in inducing marginalzone cells to initiate gastrulation movements. The most stronglyinduced part of the marginal zone begins gastrulation first(the dorsal lip of the blastopore) and proceeds through a seriesof cell interactions leading to its determination as the anteriordorsal mesoderm of the embryo. If these cell movements are inhibitedin the gastrula stage, or if vegetal induction is inhibitedin the blastula stage, or if cortical rotation is inhibitedin the first cell cycle after fertilization, the embryo alwaysfails to develop dorsal structures of the anterior end of itsbody axis; the more inhibition, the more posterior is the levelof truncation, until a radial ventralized embryo develops, derivedfrom the animal-vegetal organization of the oocyte.     

Production of hyperdorsal larvae by exposing uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole

Exposure of uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole produces larvae with enhanced dorsal structures, which resemble 'hyperdorso-anterior' larvae produced by D₂O-treatment at 0.3 normalized time (NT). Optimal conditions are 70 g for 6 min at 20% of the first cell cycle (0.2 NT). Exposure before removal of vegetal pole cortical cytoplasm, which we find has an effect of eliminating dorsal structures, protects eggs from losing their ability to form dorsal axial structures upon removal. In contrast, exposure after a slight ultraviolet (UV)-irradiation, which has virtually no effect on dorsal development, produces larvae with heavily reduced dorsal structures, which resemble 'ventralized' larvae produced by heavy UV-irradiation. Interestingly, none of these treatments prevents cortical rotation. Morphological and histological examinations reveal that exposure to the force causes displacement of both cortical and deep egg components from around the vegetal pole to subequatorial regions. We conclude that exposure to the centrifugal force enhances dorsal structures by displacing dorsal determinants from around the vegetal pole to subequatorial regions broader than normal. This is the first experiment in which displacement of egg components, by methods independent of the rotation, are shown to perturb larval body pattern.     

Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis

To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.     

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.     

Ectopic expression of Xenopus noggin RNA induces complete secondary body axes in embryos of the direct developing frog Eleutherodactylus coqui

We tested the effects of noggin RNA from Xenopus laevis on axis induction in embryos of a direct developing frog, Eleutherodactylus coqui. We microinjected noggin RNA into one blastomere of 4-cell embryos at the site close to the animal pole, and found that overexpression of noggin RNA is not only sufficient to induce additional axes but also induces heads with eyes. We also injected noggin RNA into 8-cell or 16-cell embryos in various sites, including the marginal zone, above the marginal zone, and the vegetal pole, and found the formation of a complete secondary axis in all three types of injection. These effects of X. laevis noggin RNA on the E. coqui embryo are remarkably different from those found in the X. laevis embryo itself. It has been shown previously that overexpression of noggin RNA on the ventral side of the normal X. laevis embryo induces only a partial axis, with no head structures. We show here that the failure of noggin induction of a complete axis when overexpressed on the ventral side of the X. laevis embryos is not due to an insufficient amount of RNA injected. Also, the failure is unlikely due to inhibition from the primary axis since noggin RNA can induce duplicated head structures on opposite sides of UV-irradiated X. laevis embryos. There appear to be fundamental differences in the responses of E. coqui and X. laevis embryos to exogenous noggin RNA. We propose that these differences stem from an alteration in cytoplasmic arrangements that occurred during evolution of this large egg. Received: 26 July 1999 / Accepted: 1 September 1999     

Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product

Drosophila females that lack Toll gene activity produce dorsalized embryos, in which all embryonic cells behave like the dorsal cells of the wild-type embryo. Injection of wild-type cytoplasm into young Toll- embryos restores their ability to produce a normal dorsal-ventral pattern in a position-dependent manner. No matter where the cytoplasm is injected relative to the dorsal-ventral axis of the egg shell, the position of the injected cytoplasm defines the ventralmost part of the rescued pattern. Although injection of wild-type cytoplasm into mutants at six other dorsal-group loci also restores the ability to produce lateral and ventral structures, only Toll- embryos lack any residual dorsal-ventral polarity. Experiments suggest that the activity of the Toll product is normally regulated by other dorsal-group genes and that the function of the Toll product is to provide the source for a morphogen gradient in the dorsal-ventral axis of the wild-type embryo.     

Boundaries and functional domains in the animal/vegetal axis of Xenopus gastrula mesoderm

Patterning of the Xenopus gastrula marginal zone in the axis running equatorially from the Spemann organizer-the so--called "dorsal/ventral axis"--has been extensively studied. It is now evident that patterning in the animal/vegetal axis also needs to be taken into consideration. We have shown that an animal/vegetal pattern is apparent in the marginal zone by midgastrulation in the polarized expression domains of Xenopus brachyury (Xbra) and Xenopus nodal-related factor 2 (Xnr2). In this report, we have followed cells expressing Xbra in the presumptive trunk and tail at the gastrula stage, and find that they fate to presumptive somite, but not to ventrolateral mesoderm of the tailbud embryo. From this, we speculate that the boundary between the Xbra- and Xnr2-expressing cells at gastrula corresponds to a future tissue boundary. In further experiments, we show that the level of mitogen-activated protein kinase (MAPK) activation is polarized along the animal/vegetal axis, with the Xnr2-expressing cells in the vegetal marginal zone having no detectable activated MAPK. We show that inhibition of MAPK activation in Xenopus animal caps results in the conversion of Xnr2 from a dorsal mesoderm inducer to a ventral mesoderm inducer, supporting a role for Xnr2 in induction of ventral mesoderm.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

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.