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.     

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.     

Fates and Roles of the Presumptive Organizer Region in the 32-cell Embryo in Normal Development of Xenopus laevis

Using 32-cell Xenopus embryos series of extirpation experiments were performed in order to clarify whether or not the dorsal equatorial blastomeres were committed to differentiate to the axial mesodermal structures. First, these blastomeres designated as B1, B1', C1 and C1' and C1' were labeled using the technique of HRP injection or vital staining. They produce descendants which become localized in the organizer region of the early gastrula. These cells form the prechordal plate, notochord, somites, pharyngeal endoderm and neural tube at early neurula stage. The results of extirpation of the medial two or four of these blastomeres show that the entire head lacks or the tissues and organs of the head greatly reduce. This indicates that already at the 32-cell stage they have been committed to autonomously differentiate to form the axial mesodermal tissues of the head and that their roles in the head formation can neither be replaced nor complemented by any other blastomeres surrounding them. It is also shown that the vegetal yolk cells do not seem to play essential roles for development of the axial organs of the head. On the basis of the present results a view of establishment of the organizer of Xenopus eggs is proposed.     

A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis

In Xenopus laevis, dorsal cells that arise at the future dorsal side of an early cleaving embryo have already acquired the ability to cause axis formation. Since the distribution of cytoplasmic components is markedly heterogeneous in an egg and embryo, it has been supposed that the dorsal cells are endowed with the activity to form axial structures by inheriting a unique cytoplasmic component or components localized in the dorsal region of an egg or embryo. However, there has been no direct evidence for this. To examine the activity of the cytoplasm of dorsal cells, we injected cytoplasm (dorsal cytoplasm) from dorsal vegetal cells of a Xenopus 16-cell embryo into ventral vegetal cells of a simultaneous recipient. The cytoplasm caused secondary axis formation in 42% of recipients. Histological examination revealed that well-developed secondary axes included notochord, as well as a neural tube and somites. However, injection of cytoplasm of ventral vegetal cells never caused secondary axis and most recipients became normal tailbud embryos. Furthermore, about two-thirds of ventral isolated halves injected with dorsal cytoplasm formed axial structures. These results show that dorsal, but not ventral, cytoplasm contains the component or components responsible for axis formation. This can be the first step towards identifying the molecular basis of dorsal axis formation.     

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.     

Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish.

We have analyzed the role of the zebrafish yolk cell in the processes of mesoderm induction and establishment of the organizer. By recombining blastomere-free yolk cells and animal cap tissue we have shown that the yolk cell itself can induce mesoderm in neighboring blastomeres. We further demonstrate the competence of all blastomeres to form mesoderm, suggesting the endogenous mesoderm inducing signal to be locally restricted. Ablation of the vegetal third of the yolk cell during the first 20 min of development does not interfere with mesoderm formation in general, but results in completely ventralized embryos. These embryos lack the notochord, neuroectoderm, and the anterior-most 14-15 somites, demonstrating that the ablation affects the formation of the trunk-, but not the tail region of the embryo. This suggests the presence of a trunk organizer in fish. The dorsalized mutant swirl (zbmp-2b) shows expanded dorsal structures and missing ventral structures. In contrast to the phenotypes obtained upon the ablation treatment in wild-type embryos, removal of the vegetal-most yolk in swirl mutants results in embryos which do form neuroectoderm and anterior trunk somites. However, both wild-type and swirl mutants lack a notochord upon vegetal yolk removal. These ablation experiments in wild-type and swirl mutant embryos demonstrate that in zebrafish dorsal determining factors originate from the vegetal part of the yolk cell. These factors set up two independent activities: one induces the notochord and the other is involved in the formation of the neuroectoderm and the trunk region by counteracting the function of swirl. In addition, these experiments show that the establishment of the anteroposterior axis is independent of the dorsoventral axis.     

Regional specification within the mesoderm of early embryos of Xenopus laevis

We have further analysed the roles of mesoderm induction and dorsalization in the formation of a regionally specified mesoderm in early embryos of Xenopus laevis. First, we have examined the regional specificity of mesoderm induction by isolating single blastomeres from the vegetalmost tier of the 32-cell embryo and combining each with a lineage-labelled (FDA) animal blastomere tier. Whereas dorsovegetal (D1) blastomeres induce 'dorsal-type' mesoderm (notochord and muscle), laterovegetal and ventrovegetal blastomeres (D2-4) induce either 'intermediate-type' (muscle, mesothelium, mesenchyme and blood) or 'ventral-type' (mesothelium, mesenchyme and blood) mesoderm. No significant difference in inductive specificity between blastomeres D2, 3 and 4 could be detected. We also show that laterovegetal and ventrovegetal blastomeres from early cleavage stages can have a dorsal inductive potency partially activated by operative procedures, resulting in the induction of intermediate-type mesoderm. Second, we have determined the state of specification of ventral blastomeres by isolating and culturing them in vitro between the 4-cell stage and the early gastrula stage. The majority of isolates from the ventral half of the embryo gave extreme ventral types of differentiation at all stages tested. Although a minority of cases formed intermediate-type and dorsal-type mesoderms we believe these to result from either errors in our assessment of the prospective DV axis or from an enhancement, provoked by microsurgery, of some dorsal inductive specificity. The results of induction and isolation experiments suggest that only two states of specification exist in the mesoderm of the pregastrula embryo, a dorsal type and a ventral type. Finally we have made a comprehensive series of combinations between different regions of the marginal zone using FDA to distinguish the components. We show that, in combination with dorsal-type mesoderm, ventral-type mesoderm becomes dorsalized to the level of intermediate-type mesoderm. Dorsal-type mesoderm is not ventralized in these combinations. Dorsalizing activity is confined to a restricted sector of the dorsal marginal zone, it is wider than the prospective notochord and seems to be graded from a high point at the dorsal midline. The results of these experiments strengthen the case for the three-signal model proposed previously, i.e. dorsal and ventral mesoderm inductions followed by dorsalization, as the simplest explanation capable of accounting for regional specification within the mesoderm of early Xenopus embryos.     

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

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.     

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

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 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.     

Mesodermal and axial determinants contribute to mesoderm regionalization in <Emphasis Type="Italic">Bufo arenarum</Emphasis> embryos

The existence of mesodermal determinants in the equator of Bufo arenarum embryos has been previously demonstrated. In this work, their role in dorso-ventral regionalization of mesoderm was studied by transferring the determinants to animal blastomeres. The transfer was performed by cleavage reorientation and cytoplasmic microinjection. Forced inclination during early cleavage caused deviation of the third cleavage plane and annexation of equatorial cytoplasm into animal quartets. Animal blastomeres from embryos oriented with the dorsal side up, incorporated ventro-equatorial cytoplasm and formed blood cells, mesenchyme, and coelomic epithelium. In contrast, animal blastomeres from embryos oriented with the ventral side up, acquired dorso-equatorial cytoplasm and developed notochord, somites, mesenchyme, coelomic epithelium and nervous tissue. In order to investigate if this dorso-ventral differentiation pattern responds to an interaction of mesodermal and axial factors, isolated 8-cell-stage animal quartets were microinjected with subcortical cytoplasm from: (a) the ventro-equatorial region of synchronous embryos; (b) the vegetal pole of uncleaved eggs; (c) a combination of both cytoplasms. As expected, the implanted ventro-equatorial cytoplasm promoted ventral mesoderm differentiation. Conversely, the joint transfer of ventro-equatorial cytoplasm and vegetal pole cytoplasm behaved as the dorso-equatorial cytoplasm, promoting dorso-lateral mesoderm and neural formation. Thus, mesoderm regionalization in B. arenarum embryos seems to be caused by a concurrent action of both mesodermal and axial determinants.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

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.     

Cell lineage analysis of neural induction: origins of cells forming the induced nervous system

Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.     

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.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Regional differences of proteins in isolated cells of early embryos of Xenopus laevis

Regional differences of proteins were studied by two-dimensional gel electrophoresis in early embryos of Xenopus laevis. Pairs of blastomeres on the dorso-ventral axis were isolated from 16- and 32-cell embryos. Some dorso-ventral differences have been detected at 32-cell embryos. The proteins which were clearly detectable in the vegetal cells of the ventral marginal zone were only faintly detectable or undetectable in those of the dorsal marginal zone, and a regionally specific spot was detected in dorsal blastomeres.     

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。