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.     

STUDIES ON THE FORMATION AND STATE OF DETERMINATION OF THE TRUNK ORGANIZER IN THE NEWT, CYNOPS PYRRHOGASTER

Mesoderm formation in the presumptive trunk organizer was analyzed in gastrulae of Cynops pyrrhogaster. The presumptive trunk organizer showed little or no mesodermal differentiation in the beginning gastrula (0 h embryo). But as soon as the presumptive trunk organizer came into contact with the newly invaginated cranial archenteron roof, it rapidly formed mesoderm. This suggests that this differentiation was brought about by an inductive effect of the underlying cranial archenteron roof. For investigation of this possibility, the presumptive trunk organizer of 0 h embryos (Tr-0) and the newly invaginated cranial archenteron roof (presumptive pharyngeal endoderm and prechordal plate) of successive stages were cultured in isolation and by the sandwich technique. The newly invaginated presumptive pharyngeal endoderm and prechordal plate had no effect on mesoderm formation of the presumptive trunk organizer, and mesodermal differentiation of the combinations was similar to that of the Tr-0 alone. On the other hand, results showed that the prechordal plate, which came into contact with the still uninvaginated presumptive trunk organizer, stimulated dorsalisation of the weakly mesodermized trunk organizer. Based on these results, the stepwise process of mesoderm formation in the trunk organizer is discussed.     

The role of the Spemann organizer in anterior-posterior patterning of the trunk

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.     

In Vitro Control of the Embryonic Form of Xenopus laevis by Activin A: Time and Dose-Dependent Inducing Properties of Activin-Treated Ectoderm

The inducing properties of activin-treated ectoderm of Xenopus laevis were examined by the preculture and sandwich culture methods. Presumptive ectodermal sheets of the late blastula were treated with 10–100 ng/ml of activin A and precultured for 0–7 hr in Steinberg's solution. They were then sandwiched between two sheets of ectoderm from other late blastulae. Ectoderm precultured for a short term induced trunk-tail structures, whereas that precultured for a long term induced head structures in addition to trunk-tail structures. These time-dependent changes in inducing properties occurred more rapidly when the concentration of activin A was higher. These results suggest that the activin-treated ectoderm functioned as a "head organizer" or "trunk-tail organizer" depending upon the concentration of activin A and the duration of preculture.
To trace the cell lineage of the sandwich explants, activin-treated ectoderm labeled with fluorescein-dextran-amine (FDA) was used in this study. The explants sandwiching the long term-precultured ectoderm formed head structures equipped with non-labeled neural tissues (brain and eye) as well as FDA-labeled mesodermal tissues. These results suggest that the activin-treated ectoderm mainly differentiates into mesodermal tissues and induces neural tissues as the organizer does in normal development.     

Studies on the Formation and State of Determination of the Trunk Organizer in the Newt, Cynops Pyrrhogaster III. Tangential Induction in the Dorsal Marginal Zone

Analysis of the course of differentiation of combinants between the presumptive prechordal plate (PcP) and presumptive ectoderm (PE) by time-lapse filming showed that the PcP of early gastrulae has the capacity to induce mesoderm (notochord, muscle cells and migrating cells) in the PE. The mesoderm-inducing capacity of the PcP decreases sharply during gastrulation. Following invagination in the mid-gastrula, the PcP completely loses its mesoderm-inducing capacity. This change also occurred when the PcP of the earliest gastrula was aged in vitro for 18 hr. This shows that the mesoderm-inducing capacity of the PcP decreases autonomously with aging.
PE transplanted into the presumptive trunk organizer region of the dorsal marginal zone of the earlist gastrula, became mesodermized within 12 hr. It is clear that this mesodermization of the transplanted PE is due to "tangential induction" from the PcP. The stepwise formation of the trunk organizer in Cynops pyrrhogaster is discussed in consideration of these results.     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

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.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

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).     

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.     

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.     

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.     

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.     

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.     

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

Inducing effects of the grey crescent region of early developmental stages of ambystoma mexicanum

Summary Parts of the grey crescent region of 1–2 cell, 8 cell and morula stages ofAmbystoma mexicanum were combined with presumptive gastrula ectoderm of the same species (sandwich method).Grey crescent material of the early cleavage stages induced neural tissues at a very low rate (6%–7%).From the morula stage onwards, the inducing ability of the grey crescent area increased and led to the formation of mesodermal as well as neural organs. The implanted area did not participate in the organ formations.

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Zusammenfassung Von Axolotl-Keimen wurden im 1–2 Zell-Stadium, im 8 Zell-Stadium und im Morula-Stadium Implantate aus der Region des grauen Halbmondes entnommen und mit präsumptivem Ektoderm aus frühen Gastrulen der gleichen Spezies kombiniert (Sandwich-Methode).Material aus dem grauen Halbmond früher Furchungsstadien induzierte nur wenig neurales Gewebe (6%–7% der Fälle).Vom Morula-Stadium an war die Induktionsrate höher. Neben neuralen wurden auch mesodermale Organe induziert. — Das implantierte Gewebe war nicht an den Organbildungen beteiligt.

     

Mesoderm formation in the anuranXenopus laevis (Daudin)

Summary UsingXenopus blastulae of stage 9^–, recombinates were made of the animal, ectodermal cap (zones I.II) and the vegetative, endodermal yolk mass (zone IV) (see Fig. 1). For the experiments either the entire ectodermal cap (A.B), the single outer layer (A) or the stratified inner layer (B) were used.A comparison of the quantitative composition of the recombinates and the corresponding isolates—on the basis of absolute values expressed in units of section surface area—demonstrates unequivocally that the entire mesoderm originates from the ectodermal half of the anuran egg under an inductive influence emanating from the endodermal half. This holds for recombinates of the vegetative yolk mass with the entire ectodermal cap as well as with its outer or inner layer alone.A comparison of mesoderm formation in recombinates of the entire ectodermal cap or with its outer or inner layer with the vegetative yolk mass shows that in all cases mesoderm formation is proportional to the amount of ectoderm available. In addition, the outer layer of the ectoderm is partially endodermized which may be brought in relation with the fact that in normal development an endodermal lining extends upwards from the endodermal mass, which, among other things, covers the prechordal mesoderm on the outside.The outer layer of the ectoderm has markedly lower neural competence than the inner layer, from which in normal development the bulk of the neural material arises.     

Inductive and axial properties of prospective wing-bud mesoderm in the chick embryo

Prospective wing-bud mesoderm, stripped of ectoderm mechanically through the use of glass needles, or chemically by means of EDTA or trypsin, was obtained from donor embryos of stages 11 through 21. Grafts were made in both homopleural (aadd and apdv) and heteropleural (aadv and apdd) combinations to the right flank of host embryos of the same range of stages. Flank ectoderm from the host healed over the graft in a few hours and, in combinations between donors and hosts in the range of stages 12 through 17, the composite formed, with high frequency, a limb bud capped by an apical ectodermal ridge, and then developed into a supernumerary wing in which all proximodistal levels were represented. When either member of the combination was older than stage 17, only incomplete limbs, if any, were formed. Regardless of their orientation on the host, the supernumerary limbs always showed the axial characteristics appropriate to their side of origin.Supernumerary wings failed to form if the grafts were inserted into a space tunneled between flank ectoderm and its underlying mesoderm. If the covering ectoderm were deliberately torn and forced to heal over the graft, however, an ectodermal ridge formed and a supernumerary limb developed.It is concluded, therefore, that: (1) the wing-bud mesoderm, appropriately combined with flank ectoderm, has the property of morphological and axial self-differentiation by stage 12; (2) the apical ectodermal ridge is induced in flank ectoderm by prospective wing-bud mesoderm; (3) this inductive power is restricted to prospective wing-bud mesoderm from donors of stages 12 through 17; (4) the response competence is limited to flank ectoderm that has healed over the mesoderm; and (5) this competence is lost by the end of stage 17.