Head and trunk-tail organizing effects of the gastrula ectoderm of Cynops pyrrhogaster after treatment with activin A

Differentiation tendency and the inducing ability of the presumptive ectoderm of newt early gastrulae were examined after treatment with activin A at a high concentration (100 ng/ml). The activin-treated ectoderm differentiated preferentially into yolk-rich endodermal cells. Combination explants consisting of three pieces of activin-treated ectoderm formed neural tissues and axial mesoderm along with endodermal cells. However, the neural tissue was poorly organized and never showed any central nervous system characteristics. When the activin-treated ectoderm was sandwiched between two sheets of untreated ectoderm, the sandwich explants differentiated into trunk-tail or head structures depending on the duration of preculture of activin-treated ectoderm in Holtfreter's solution. Short-term (0–5 h) precultured ectoderm induced trunk-tail structures accompanied by axial organs, alimentary canal and beating heart. The arrangement of the explant tissues and organs was similar to that of normal embryos. However, archencephalic structures, such as forebrain and eye, were lacking or deficient. On the other hand, long-term (10–25 h) precultured ectoderm induced archencephalic structures in addition to axial organs. Lineage analysis of the sandwich explants using fluorescent dyes revealed that the activin-treated ectoderm mainly differentiated into endodermal cells and induced axial mesoderm and central nervous system in the untreated ectoderm. These results suggest that activin A is one of the substances involved in triggering endodermal differentiation and that the presumptive ectoderm induced to form endoderm displays trunk-tail organizer or head organizer effects, depending on the duration of preculture.     

Role of BMP-4 in the inducing ability of the head organizer in Xenopus laevis

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP-4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.     

Activin-treated ectoderm has complete organizing center activity in Cynops embryos

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops .     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

EFFECT OF AGING ON NEURAL COMPETENCE OF PRESUMPTIVE ECTODERM, AND EFFECT OF AGED ECTODERM ON DIFFERENTIATION OF THE ORGANIZER IN CYNOPS PYRRHOGASTER II. ROLE OF EXTREME POSTERIOR OF THE ARCHENTERIC ROOF IN THE SLIT-BLASTOPORE STAGE IN NORMAL DEVELOPMENT

The effect of aging on the neural competence of the presumptive ectoderm in gastrulae of Cynops pyrrhogaster and the effect of aged ectoderm on differentiation of the extreme posterior of the archenteric roof in the slit-blastopore stage were examined by a sandwich method in which this organizer was wrapped in the presumptive ectoderm taken from the 0- to 42-hr aged exogastrulae. Vital staining showed that this organizer becomes mainly tail notochord. Therefore it should be called tail or trunk-tail organizer. In 0- to 18-hr explants, typical trunk-tail structures were formed. With further aging of the presumptive ectoderm, a decrease of spinal cord and muscle with a concomitant increase of mesenchyme and mesothelium was observed. In 36- (corresponding to the slit-blastopore-initial neural stage) and 42-hr explants, neural competence had disappeared markedly. The notochord appeared in all explants, indicating this organizer is more firmly determined than the uninvaginated dorsal lip in small yolk-plug stage. Conclusively, this organizer does not play an important role in the induction of the neural plate, but induces the tail in normal development.     

Mesoderm and Neural Inductions on Newt Ectoderm by Activin A

Mesoderm-inducing activity of human recombinant activin A was examined on presumptive ectoderm of the Japanese newt, Cynops pyrrhogaster , by using the animal cap assay, Activin A induced neural tissues and mesodermal tissues such as brain, neural tube, notochord, muscle, mesenchyme, coelomic epithelium and blood-like cells after 14 days cultivation. These tissues were induced by activin A at concentrations ranging from 0.5– 100 ng/ml. Dose-dependent inducing activity of activity A on newt ectoderm was slightly different from that on other animals, including Xenopus . Wide range of concentration of activin A (0.5– 100 ng/ml) could induce the neural tube, notochord, mesenchyme and coelomic epithelium on the newt ectoderm. Though the percentage of induced explants (two out of 23 explants, 8.7%) was low, the pulsating heart was induced. This paper showed first that activin could induce the mesodermal and neural tissues in newt presumptive ectoderm. Since activin homologues were present In Xenopus and chick embryos, it is likely that activin may be one of the natural inducers in a wide range of species.     

Spemann's influence on Japanese developmental biology

The discovery of the organizer by H. Spemann and Hilde Mangold, prompted a number of studies of embryonic induction in Japan. C.O. Whitman, N. Yatsu, T. Sato, H. Oka, T. Yamada, and Y.K. Okada were the pioneers in the field of embryonic induction. T. Yamada postulated the double potential theory for embryonic induction. O. Nakamura has modified the fate map of Vogt using newt and Xenopusblastulae. T.S. Okada and G. Eguchi proposed the new concept of "transdifferentiation" based on in vitro experiments in the retina and lens. T.S. Okada is not only an excellent scientist, but he has also nurtured many active developmental biologists. M. Takeichi, from his school, discovered the cell adhesion molecle, cadherin. Nakamura and colleagues tried to determine the origin and formation of the organizer. They performed recombination experiments using the ectoderm, endoderm and mesoderm, and concluded that the phenomenon in which various mesoderm tissues are formed by the recombination of the presumptive ectoderm with endoderm was "regulation of the vegetal-animal gradient". Some groups have also tried to purify specific inducing factors. T. Yamada and colleagues isolated two different types of ribonucleoproteins. I. Kawakami and colleagues showed that the ribosome fraction has neural inducing capacity, and that the extracellular matrix contains mesodermal inducing factors. Finally Asashima and colleagues isolated and identified activin A as a MIF factor. This finding had a great influence not only in the field of developmental biology, but also in molecular biology. Using activin, Asashima's group has successfully generated various organs, tissues, trunk-tail and head structures in vitro using animal caps (undifferentiated cells). Some other important molecules such as BMP, chordin and bFGF are also being studied by young Japanese scientists.     

BRAIN INDUCTION IN VARIOUSLY AGED PRESUMPTIVE ECTODERMS BY HEAD ORGANIZER IN CYNOPS PYRRHOGASTER

Brain formation in variously aged presumptive ectoderms of Cynops pyrrhogaster under the influence of the head organizer was examined by the sandwich method. The head organizer was obtained from the middle portion of the archenteron roof at the slit-blastopore stage. The presumptive ectoderm was taken from 0- to 36-hr exogastrulae. Exogastrulae were prepared from the earliest gastrulae just before invagination (0-hr embryos). The presumptive neural plate overlying the archenteron roof used as organizer was cultivated in an envelope of belly ectoderm from an early neurula.
The following results were obtained: 1) Brain induction was almost entirely restricted to explants covered with 6-hr ectoderm and its frequency was low. 2) The presumptive neural plate above the head organizer was almost completely determined as neural tissues. 3) The head organizer showed a tendency to differentiate into more endodermal and less mesodermal tissues than those expected from its prospective fate.
Brain induction in normal development and the relationship between neural tissue formation in variously aged presumptive ectoderms and the time necessary for neural induction are discussed.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

INDUCING TIME FOR NEURAL TISSUES IN GASTRULA ECTODERM OF CYNOPS PYRRHOGASTER

The inducing times for spinal cord and deuterencephalon in Cynops gastrula were determined by the sandwich method. The extreme posterior of the archenteron roof at the slit-blastopore stage (tail organizer) was used as an inducer. First, the presumptive ectoderm of the earliest gastrula (0-hr stage) was put in contact with the organizer for 6 to 24 hr. Spinal cord and deuterencephalon were induced in almost all explants after 24 and 21 hr of contact, respectively, indicating that 24 hr is enough time for differentiations of both spinal cord and deuterencephalon. Next, presumptive ectoderm of 6- to 21-hr exogastrulae was put in contact with the organizer until the 24-hr stage. Results showed that the net inducing times for spinal cord and deuterencephalon were 18 and 15 hr, respectively, and that neural competence appeared in the presumptive ectoderm at the 6-hr stage (straight-blastopore stage).     

Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.     

Epithelial Cell Wedging and Neural Trough Formation Are Induced Planarly inXenopus,without Persistent Vertical Interactions with Mesoderm

In this study we investigate the induction of the cell behaviors underlying neurulation in the frog,Xenopus laevis.Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Kelleret al.,1992,Dev. Dyn.193, 218–234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a “giant sandwich” explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.     

Inductive Capacities for the Dorsal Mesoderm of the Dorsal Marginal Zone and Pharyngeal Endoderm in the Very Early Gastrula of the Newt,and Presumptive Pharyngeal Endoderm as an Initiator of the Organization Center*

The organization center of Cynops pyrrhogaster was divided into Parts 1, 2 and 3 of equal size (0.3×0.4 mm²) with presumptive fates as pharyngeal, pharyngeal+prechordal+trunk notochord, and trunk-tail notochord, respectively. Movements and changes in size and shape of each part were followed through gastrulation. Differentiation tendencies of each part were examined under three conditions: I, isolated; II, sandwiched with presumptive ectoderm; 111, sandwiched with presumptive ectoderm after preculture in isolation for various times. In I, Parts 2 and 3 differentiated into dorsal mesoderm. In II, each part induced dorsal mesoderm and neural tissues, the frequency being highest in Part 2 and lowest in Part 3. In III, Parts 1 and 2 realized their presumptive fates, through changes in inductive capacities from trunk-tail to head. This change progressed rapidly in Part 1, and slowly in Part 2. Part 3 required induction by neighbouring Part 2 to realize its presumptive fate. Changes of inductive capacity of Parts 1 and 2 respectively, were chronologically similar in normal development and in preculture experiments. Lastly, the primary presumptive pharyngeal zone at blastula was proposed to act as an initiator of the organization center, its programmed information being transmitted to Part 2, and then to Part 3.     

The effect of aging on the neural competence of the presumptive ectoderm and the effect of aged ectoderm on the differentiation of the trunk organizer inCynops pyrrhogaster

Summary The effect of aging on the neural competence of the presumptive ectoderm of the early gastrula, and the effect of aged ectoderm on the differentiation of the still uninvaginated dorsal blastoporal lip at the small yolk-plug stage — representing the trunk organizer — were examined by the sandwich method inCynops pyrrhogaster.The presumptive ectoderm to be used as reaction system was taken from 0 to 36 h exogastrulae obtained by operation at the early gastrula stage and combined with trunk organizer. In the 0 to 12 h explants typical trunktail structures were formed. With further aging of the presumptive ectoderm a decrease in frequency of spinal cord, notochord, and muscle and a simultaneous increase in frequency of mesenchyme and mesothelium were observed. In the 30 and 36 h explants neural competence had largely disappeared, the frequency of notochord and muscle become very low and their differentiation very poor, whereas the frequency of mesenchyme and mesothelium reached very high levels.We infer a reciprocal relationship between the induced spinal cord and the differentiation of notochord and muscle, as well as a transformation of notochordal material into mesenchyme and mesothelium under the influence of the aged ectoderm. The mode of action of the trunk organizer in normal development is discussed.     

Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin.

The peptide growth factor Activin A has been shown to induce complete axial structures in explanted blastula animal caps. However, it is not understood how much this response to activin depends upon early signals that prepattern the ectoderm. We have therefore asked what tissues can be induced in blastula animal caps by activin in the absence of early dorsal signals. Using whole-mount in situ hybridization, we compare the expression of three neural markers, N-CAM, En-2 and Krox-20 in activin-treated ectoderm from control and ventralized embryos. In response to activin, both normal and ventralized animal caps frequently form neural tissue (and express N-CAM) and express the hindbrain marker Krox-20. However, the more anterior marker, En-2, is expressed in only a small fraction of normal animal caps and rarely in ventralized animal caps; the frequency of expression does not increase with higher doses of activin. In all cases En-2 and Krox-20 are expressed in coherent patches or stripes in the induced caps. Although mesoderm is induced in both control and ventralized animal caps, notochord is found in response to activin at moderate frequency in control caps, but rarely in ventralized animal caps. These results support the idea that in the absence of other signals, activin treatment elicits hindbrain but not notochord or anterior neural tissue; and thus, the anterior and dorsal extent of tissues formed in response to activin depends on a prior prepatterning or previous inductions.     

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

In vitro control of organogenesis and body patterning by activin during early amphibian development

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.     

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.