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 Location of Dorsal Information in Frog Early Development

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.     

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。     

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.     

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.     

Onset of competence to respond to activin A in isolated eight-cell stage Xenopus animal blastomeres

Single animal hemisphere blastomeres isolated from the eight-cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32-cell stage) of normal development. The competence gradually increased during the morula stages.     

Spatial and temporal properties of ventral blood island induction in Xenopus laevis.

Questions of dorsoventral axis determination and patterning in Xenopus seek to uncover the mechanisms by which particular mesodermal fates, for example somite, are specified in the dorsal pole of the axis while other mesoderm fates, for example, ventral blood island (VBI), are specified at the ventral pole. We report here that the genes Xvent-1, Xvent-2, and Xwnt-8 do not appear to be in the pathway of VBI induction, contrary to previous reports. Results from the selective inhibition of bone morphogenetic protein (BMP) activity, a key regulator of VBI induction, by ectopic Noggin, Chordin, or dominant negative BMP ligands and receptors suggest an alternative route of VBI induction. Injection of noggin or chordin RNA into animal pole blastomeres effectively inhibited VBI development, while marginal zone injection had no effect. Cell autonomous inhibition of BMP activity in epidermis with dominant negative ligand dramatically reduced the amount of (&agr;)T3 globin expression. These results indicate that signaling activity from the Spemann Organizer alone may not be sufficient for dorsoventral patterning in the marginal zone and that an inductive interaction between presumptive VBIs and ectoderm late in gastrulation may be crucial. In agreement with these observations, other results show that in explanted blastula-stage marginal zones a distinct pattern develops with a restricted VBI-forming region at the vegetal pole that is independent of the patterning activity of the Spemann Organizer.     

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.     

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.     

Activin-like signal activates dorsal-specific maternal RNA between 8- and 16-cell stages of Xenopus

In many animals the dorsalventral axis forms by an initial localization of maternal molecules, which then regulate the spatial location of signals that directly influence the expression of axis-specific fates. Several recent studies have demonstrated that dorsal-animal blastomeres of the Xenopus morula (8–32 cells) are biased toward dorsal fates prior to mesoderm inductive signaling In this study we ask whether the dorsal bias is the result of autonomous expression of maternal molecules specifically localized within dorsal cells or of early activating signals. It was found that although 16-cell dorsal-animal blastomeres (D1.1) can differentiate into dorsal tissues when cultured alone, the 8-cell mothers (D1) can not. Likewise, although RNA extracted from D1.1 can induce an extra dorsal axis when injected into vegetal blastomeres, RNA extracted from D1 can not. However, D1 does express dorsal tissues if co-cultured with dorsal-vegetal cells or with culture medium containing a mixture of activins (PIF-medium). Furthermore, short-term culture of D1 in PIF-medium enables the D1 RNA to induce an ectopic dorsal axis. Ven ral-animal blastomeres also can express dorsal axial tissues when co-cultured with dorsal-vegetal blastomeres or in PIF-medium, but the RNA from the activin-treated ventral cells cannot induce ectopic dorsal axes. These studies demonstrate that there are maternal RNAs that, shortly after fertilization are present only in the dorsalanimal region. They do not act cell autonomously, but require an activin-like signal. These RNAs may function by increasing the responsiveness of dorsal-animal blastomeres to the mesoderm inductive signals present in both the morula and the blastula. © Wiley-Liss, Inc.     

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.     

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.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). "Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus." Development. 126, 3703-3713.]. We present a revised model of gastrulation movements in Xenopus laevis.     

Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus.

An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.     

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.     

Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.

Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.     

Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg

The molecular basis for the initial specification of dorsoventral polarity in the Amphibian egg prior to the mid-blastula transition still remains an open question. Regional differences in the protein pattern of Pleurodeles egg were investigated during early cleavage (8- and 512-cell stages, prior to the mid-blastula transition). Animal-dorsal, animal-ventral, vegetal-dorsal and vegetal-ventral quarters were separated and proteins were analyzed by 2D-electrophoresis. The comparison of acidic protein patterns from dorsal and ventral quarters revealed differences between vegetal cells but no difference was detected between animal cells. One protein (p11, 30 kDa) was characterized in the dorsal side as early as the 8-cell st. and two dorsal spots were detected at the 512-cell st. (p11 and p5, 65 kDa). Similarly one protein (p7b, 46 kDa) appears to be ventral-specific from the 8-cell st. The p11 spot was shown to appear in ventral cells as a consequence of a dorsalizing LiCl-treatment at the 32-cell stage. Conversely, p11 disappeared from dorsal cells and p5 did not appear at 512-cell stage after UV-irradiation of the uncleaved egg, which results in the expression of the ventral-specific protein p7b in the dorsal part of the egg. Therefore differential protein expression is already present at very early cleavage stages. Its significance needs further investigation.