Establishment of the organizing activity of the lower endodermal half of the dorsal marginal zone is a primary and necessary event for dorsal axis formation in Cynops pyrrhogaster.

The formation of the head and trunk-tail organizers in the dorsal marginal zone (DMZ) of an amphibian embryo is thought to require spatial and temporal interactions between the Nieuwkoop center and the DMZ. Recent studies of the Xenopus embryo suggested that intra-DMZ interaction is also needed to establish the regional specificity of the DMZ. However, it is not yet clarified when and how the final pattern of the head and trunk-tail organizers is established. To analyze the intra-DMZ interactions, we injected suramin into the blastocoel of the mid-blastula of the urodele, Cynops pyrrhogaster, at 6 h prior to the onset of gastrulation. The pigmented blastopore formed normally, but the convergent extension and involution of the DMZ and dorsal axis formation of the embryo were completely inhibited. Expression of gsc, chd and Lim-1 were not maintained, but noggin was unaffected in the suramin-treated embryos. Dorsal axis formation and the expression of these genes of the suramin-treated embryos were rescued by replacing the lower endodermal half of the DMZ (LDMZ) with normal LDMZ. The present results of embryological and molecular examinations indicate that organizing activity of the early Cynops gastrula DMZ is restricted to the LDMZ, and that the organizing activity of the LDMZ is established during the late blastula stages. The results also indicate that LDMZ triggers the sequential interaction within the DMZ that establishes the final pattern of the regional specificity of the DMZ, and that the formation of the LDMZ is a primary and necessary event for dorsal axis formation.

Cellular Basis of Neuralization of Induced Neurectoderm in Amphibian Embryogenesis: Changes of Cell Shape, Cell Size, and Cytodifferentiation of the Neurectoderm after Neural Induction: (neuralization/primary embryonic induction/cellular changes/amphibia (Cynops)).

Cellular alterations of the neurectoderm after primary embryonic induction were examined by measuring several indices of shape, volume, and cytodifferentiation of the neurectodermal cells of Cynops embryos during gastrulation and early neurulation. Results showed that cellular alterations occurs just after the 18 hr embryo stage (stage 13b). The thickness of the neurectoderm layer decreases like that of the epidermal ectoderm during early and middle gastrulation. After the 18 hr embryo stage, however, the neurectoderm thickens, mainly due to formation of columnar cells. Measurement of cell volume indicates that the neurectoderm of the early and middle gastrulae consists of a cell population of heterogeneous size. The heterogeneity diminishes sharply after the 18 hr embryo stage and the neural plate of the 36 hr embryo (stage 18) consists of cells of homogeneous size. Stages before the 12 hr embryo (stage 12b) and after the 18 hr embryo (stage 13b) also showed differed in cell adhesion to the culture flask and in cytodifferentiating potency. Single cells dissociated from the neurectoderm of 18 hr embryos could adhere to the substratum and differentiate into both nerve-like cells and pigment cells. Both capacities increase during further development. These results are discussed in relation to the neuralizing determination of neurectoderm after primary embryonic induction.

Prospective Neural Areas and their Morphogenetic Movements during Neural Plate Formation in the Xenopus Embryo. II. Disposition of Transplanted Ectoderm Pieces of X. borealis Animal Cap in Prospective Neural Areas of Albino X. laevis gastrulae.: (developmental fate/neural plate area/Xenopus embryo/chimera/quinacrine).

Small pieces of the animal cap of X. borealis gastrulae were transplanted into various regions of the noninvoluting marginal zone of albino X. laevis gastrulae, and the distribution of the donor cells was analyzed by quinacrine fluorescence staining. The present study indicated that the prospective central nervous system (CNS) lies as a belt-shaped area in the noninvoluting marginal zone of early gastrulae. This belt-shaped prospective neural area extends as far as 0.7 mm (115° to the vegetal pole) above the blastopore in the dorsal midline and 1.3 mm lateral (130° to the dorsal midline) to the dorsal midline. The ectoderm of the dorsal region extends in the animal-vegetal direction and forms the ventral side of the CNS. The dorsalateral and lateral regions converge toward the dorsal midline and extended in the animal-vegetal direction. The former constitutes the lateral side of the anterior CNS, and the latter the dorso-lateral side of the posterior CNS. The outer layer of ectoderm which was transplanted onto the inner layer of the host gastrula differentiated into neural tissues. The prospective areas of the CNS and their morphogenetic movement during Xenopus embryogenesis are also discussed with regard to neural induction.

Prospective Neural Areas and Their Morphogenetic Movements during Neural Plate Formation of Xenopus Embryos. I. Development of Vegetal Half Embryos and Chimera Embryos: (developmental fates/cell marker, quinacrine/Xenopus embryo).

Development of animal cap-less Xenopus gastrulae was examined. In vegetal halves from which the animal cap was removed 0.6 mm above the blastopore, an apparently normal array of craniocaudal structures developed. Histological examination showed differentiation of central nervous system (CNS) structures in the cap-less embryos, but differentiation of sensory organs, such as a lens and ear vesicle in only a few embryos. Only the dorsal midline of the embryos was covered with epidermis, and its lateral-ventral areas consisted of bare endoderm and mesoderm. The development of animal cap was also investigated by exchanging the animal cap of X. laevis embryos with that of X. borealis embryos, which can be distinguished by quinacrine fluorescence staining. The central nervous system of chimera embryos consisted mainly of X. laevis cells stained homogeneously with quinacrine but a small number of punctately-stained X. borealis cells was in the anterior tip of the forebrain. Cells of the lens and ear vesicle were punctately stained. More than two-thirds of the epidermal area consisted of punctately-stained cells and only the dorsal midline of the posterior head- and trunk-epidermis consisted of homogeneously-stained cells. Areas of the prospective central nervous system and their movement during embryogenesis of Xenopus are discussed.

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction: (germ layer interaction/mesoderm formation/embryonic induction/amphibian (Cynops) axis).

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm. Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof. The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.

Studies on the formation and state of determination of the trunk organizer in the newt,Cynops pyrrhogaster : IV. The association of the neural-inducing activity with the mesodermization of the trunk organizer.

The stepwise process of the formation and determination of mesoderm inCynops pyrrhogaster was analyzed. The presumptive ectoderm (PE) of the early gastrula was transformed into mesoderm within 12 h when transplanted into the upper half of the dorsal marginal zone of the same stage. The self-differentiation capacity and the neural-inducing activity of this newly mesodermized PE (MPE) were examined by both isolation and sandwich cultures.The MPE showed self-differentiation for notochord and muscle in the isolation culture. In the sandwich culture, the MPE made contact with the PE of the successive gastrula stages. The MPE was capable of inducing neural tissues even in the PE of the mid-gastrula, which has high neural competence but loses it within a short period of 6 h.These results show that firstly the mesodermization of the PE is completed within 12 h and secondly both the self-differentiation capacity and the neural-inducing activity are established immediately after the mesodermization of the PE.

Neural-inducing activity and glycoprotein synthesis of newly mesodermalized ectoderm in the newtCynops (Amphibia).

An artificially mesodermalized ectoderm (mE) shows the same properties as the organizer: chordamesoderm formation and neural induction. The neural-inducing activity of the mE was inhibited by treatment with protein synthesis inhibitors (cycloheximide and puromycin) and a specific inhibitor of protein glycosylation (tunicamycin). These antibiotics also inhibited chordamesoderm differentiaton, especiallly that of notochord. Newly synthesized proteins of the mE were compared with those of presumptive ectoderm (pE) using two-dimensional PAGE. There were differences in relative amounts of many protein spots. These results suggest that neural-inducing activity is related to glycoproteins synthesized during the early phase of mesodermalization.

Two essential processes in the formation of a dorsal axis during gastrulation of Cynops embryo.

The isolated upper marginal zone from the initial stage of Cynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.

Changes in the interior surface of newly mesodermalized ectoderm and its contact activity with competent ectoderm in the newtCynops (Amphibia).

The presumptive ectoderm (pE) ofCynops gastrulae was artificially mesodermalized by contact with teleost swimbladder. The newly mesodermalized ectoderm (mE) acquired the capacity for neural induction (Suzuki et al. 1986a). SEM observations revealed that the mE cells altered their cellular profiles immediately after mesodermalization. The characteristics of the cell surface and the cell architecture became similar to those of invaginated mesoderm cells. There were distinct differences in the cellular contact between mE-pE and pE-pE combinations. The mE-pE combinations kept close contact at their interior surfaces, while the pE-pE combinations did not keep contact. Both TEM and SEM observations also indicated that there were tight contacts between mE and pE cells. These findings suggest that neural-inducing activity of the newly mesodermalized ectoderm cells is coupled with acquisition of cellular affinity toward the interior surface of competent ectoderm cells, and probably requires close cell contacts.

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis.

For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 103 and 30 × 103 Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level.

A useful approach for the screening of active neural-inducing factors.

The present study suggests that the membrane-binding molecules of mesodermal cells and/or the modulated extracellular matrix (ECM) with them play an important role in induction of the central nervous system. Artificially mesodermalized ectoderm (mE) or chordamesoderm (cM) was placed on a collagen and flbronectin (CF)-coated dish for 24 h. After mechanical removal of the mesoderm sheet, competent ectoderm of early gastrulae was placed on the same spot. Many melanocytes and neuronal cells were observed after 1 week, along with many cells which reacted specifically with a neuralspecific monoclonal antibody. However, when presumptive ectoderm (pE) instead of mE or cM was used as the control, only epidermal cells with cilia were observed in the competent ectoderm, except for a few melanocytes in rare cases. The proteins synthesized and remaining on the CF substrate during placement of the mE and pE were analysed by two-dimensional polyacrylamide gel electrophoresis (PAGE) fluorography. The fluorography indicated that there were significant differences between the polypeptides spots of mE and pE.

Possible mechanisms in the rearrangement of non-yolk cytoplasmic materials during maturation of theXenopus laevis oocyte.

Using the monoclonal antibody (MoAb) Xa5B6 as probe, the authors examined the mechanisms of cytoplasmic rearrangement occurring during maturation of theXenopus oocyte. The antigen molecules recognized by the MoAb are arranged in radial striations of the oocyte cytoplasm. The radial striations were disorganized in vitro by progesterone treatment, and the antigen molecules were uniformly distributed, predominantly in the animal hemisphere. Even when the germinal vesicle was mechanically removed or when germinal vesicle breakdown was suppressed in a K+-free medium, progesterone induced a disorganization of the radial striations. This progesterone-induced disorganization was inhibited by the protein synthesis inhibitor cycloheximide. When full-sized oocytes were treated with cytochalasin B, the radial striations were also disorganized, but the antigen molecules did not disperse into the large mass. Colchicine treatment had little effect. Antigen molecules were no longer arranged in radial striations and were completely dispersed when the oocyte was simultaneously treated with both drugs. These results indicate that the two compartments in the oocyte cytoplasm, the yolk-free cytoplasm and yolk column, are organized by different types of cytoskeletal system. It is also suggested that the maturation-promoting factor (MPF) activated during progesterone-induced maturation disrupts these cytoskeletal systems and disorganizes the radial striations.

Neural-inducing activity of newly mesodermalized ectoderm.

The neural-inducing activity of artificially mesodermalized ectoderm was examined. The competent ectoderm of earlyCynops gastrula was mesodermalized by being placed in contact withCarassius swimbladder. The mesodermalized ectoderm was combined with ectoderm isolated from various developmental stages of a gastrula. Neural differentiation were observed in half the combinants, even in 18 h ectoderm, which is considered to have lost its neural competence within 6 h. This indicates that mesodermalized ectoderm is capable of inducing neural tissues at the very time it comes into contact with 18 h ectoderm. From the present study, the neural-inducing activity of mesodermalized cells may possibly be closely connected to the early process of their mesodermalization.

Alteration of cell adhesion system in amphibian ectoderm cells during primary embryonic induction: changes in reaggregation pattern of induced neurectoderm cells and ultrastructural features of the reaggregate.

Cell adhesion was studied during primary embryonic induction. The disaggregation rate and reaggregation patterns were analysed in the ectoderm cells of various developing Cynopus gastrulae and neurulae. The neurectoderm cells disaggregated more slowly with gastrulation, and the neural plate cells of early neurula showed a lesser capacity for disaggregation. Although no differences in reaggregation were found between dorsal and ventral ectoderm at the early gastrula stage, there were significant differences between the induced neurectoderm and the non-induced ventral epidermal cells at the late gastrula stage. Neural plate cells of the early neurula stage were seen to form a chain-like reaggregate, but the ventral epidermal cells of the same embryo formed a cluster-like spherical reaggregate. Scanning electron microscope observations of reaggregates also showed significant differences in adhesive properties between induced neurectoderm and non-induced epidermal cells. The adhesion field of the induced neurectoderm cells was smooth, differing from the distinct ridges of the non-induced epidermal cells. These results suggest that changes in the cell adhesion system, resulting in the formation of a columnar cell shape, may occur immediately after a neural-inducing action.

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis.

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.

Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody.

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole-mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior-posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm-inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis.

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.

Regional specification of the head and trunk-tail organizers of a urodele (Cynops pyrrhogaster) embryo is patterned during gastrulation.

The dorsal marginal zone (DMZ) of an amphibian early gastrula is thought to consist of at least two distinct domains: the future head and trunk-tail organizers. We studied the mechanism by which the organizing activities of the lower half of the DMZ (LDMZ) of the urodelean (Cynops pyrrhogaster) embryo are changed. The uninvoluted LDMZ induces the notochord and then organizes the trunk-tail structures, whereas after cultivation in vitro or suramin treatment, the same LDMZ loses the notochord-inducing ability and organizes the head structures. A cell-lineage experiment indicated that the change in the organizing activity of the LDMZ was reflected in the transformation of the inductive ability: from notochord-inducing to neural-inducing activity. Using RT-PCR, we showed that the LDMZ expressed gsc, lim-1, chordin, and noggin, but not the mesoderm marker bra. In the sandwich assay, the LDMZ induced bra expression in the animal cap ectoderm, but the inductive activity was inhibited by cultivation or suramin treatment. The present study indicates that the change in the organizing activity of the LDMZ from trunk-tail to head is coupled with the loss of notochord-inducing activity. Based on these results, we suggest that this change is essential for the specification of the head and trunk-tail organizers during gastrulation.