Gastrula Ectoderm Research Articles

High performance gel permeation chromatography of proteins. Application to embryonic inducing factors.

Proteins in the molecular weight range of 10 000-170 000 were separated by high performance gel permeation chromatography. Silica particles with 30 nm or 50 nm pores were derivatized with glycidoxypropyltrimethoxysilane and used as support. The proteins were eluted with 50% formic acid. A protein fraction which induces endodermal and mesodermal tissues in amphibian gastrula ectoderm was purified by this method.

CHANGES IN THE ELECTROPHORETIC MOBILITY OF PRESUMPTIVE ECTODERM CELLS TREATED WITH MESODERMAL INDUCING SUBSTANCES.

Using gastrula ectoderm of Cynops pyrrhogaster, the electrophoretic mobility (EPM) of cells was measured. Before EPM measurement, the cells were treated with either LiCl or swimbladder tissue, which are known to possess endo- and mesodermal inducing activity. The EPM of the treated cells increased after 48 hr. A significant difference was observed between the control and experimental series. In addition to the measurements of EPM, histological sections of the explants were examined. Before obvious tissue differentiation occurred in the explants, definitive changes in the EPM values were recorded at 48 and 72 hr after treatment.

The formation of mesodermal derivatives after induction with vegetalizing factor depends on secondary cell interactions

Early amphibian gastrula ectoderm induced with vegetalizing factor for 6 h using the sandwich-method and cultured for up to 12 days, differentiated into mesodermal and endodermal tissues. Explants which were dissociated into single cells after the induction followed by immediate reaggregation and then cultured for 12 days likewise differentiated into mesodermal and endodermal tissues. However, if after induction and dissociation of the tissue, single cells are cultured for 20 h prior to the reaggregation, the reaggregated cell mass mainly differentiated into endoderm (liver and intestine) and irregularly shaped epidermis (formerly called ‘atypical epidermis’). Blood cells and, in few cases, heart structures were the only mesodermal structures found in this series. The results suggest that an endodermal anlage is induced first. The differentiation of mesodermal derivatives depends on secondary cell interactions between endodermal induced and non-induced ectoderm. For this process permanent cell-to-cell contacts are necessary.

Neural crest replaced by gastrula ectoderm in amphibia. Effect on neurulation, CNS, gills and limbs.

Early Axolotl gastrula ectoderm was grafted into early Triturus neural stages in place of excised neural folds at the gill and anterior trunk level. Macroscopically the young graft behaves like normal neural fold material: it follows the closing host neural plate to the dorsal midline, folds into the host's interior and, especially in the gill region, moves ventrad beneath the host's epidermis. These movements cannot be interpreted as active migration. They are the result of passive displacements by morphogenetic forces inside the embryo. Histologically the graft differentiates into neural and neural crest tissue, the quantitative relation depending on the host's region. At the gill level the graft forms mesenchyme and other neural crest elements and hardly any neural structures. In the trunk about one half of the graft forms a secondary, surplus CNS. Problems of induction, differences between gill and trunk region and between graft and normal fold behaviour are discussed. Limbs develop normally. The dorsal layer of the blastema is furnished by graft cells. Host and graft tissue can stay separate or form a combined blastema.

The Concept of Embryonic Induction in the Passage of Time

The paper critically reviews, in historical perspective, the theoretical aspects of interactions of germ layers, which control subsequent differentiation of somatic tissues in amphibian embryos. The adequacy of the classical argument which resolved the organizer action into actions of individual inducing substances is questioned, and the usefulness of the theoretical models which operate with morphogenetic events as opposed to inducing substances is emphasized. It is pointed out that the prospective ectoderm of an early amphibian gastrula has the ability to produce almost all somatic tissues belonging to the three germ layers under various epigenetic conditions, and hence omnipotency could be ascribed to it. The importance of the rather recent recognition that the developmental pathways of the mesoderm are established under the influence of the endoderm, before organizer action starts, is stressed. The influence of the endoderm is interpreted as vegetalization of the animal hemisphere. A review of the studies on some heterogenous inductors, which were originally intended to characterize the chemical factors involved in organizer action, shows that the main components of those inductors have strong vegetalizing effects on competent ectoderm. Thus the effect of those inductors and their effective molecules should be evaluated for the mechanism of vegetalization of the upper hemisphere before gastrulation, rather than for the mechanism of the organizer action itself. The regionalization of neural tissue produced by heterogenous vegetalizing inductors could be due to a secondary interaction between the ectoderm and mesoderm which has been produced by the vegetalizing effects of those inductors. If this is the case, those inductors do not give any clue as to the chemical factors involved in regionalization by the organizer in normogenesis, in contradiction to the past dominating attitude in this area. The reason that one should take into consideration a possible causal role of morphogenetic movements in the mechanism of regionalization is presented. My task is to critically review the studies of developmental interactions between germ layers of amphibian embryos leading to the appearance of somatic tissues, an area to which Pieter Nieuwkoop contributed a great deal. The focus of the paper is on the theoretical aspects of the problem as they evolved in the last three decades, and not on the experimental data, most of which have been reviewed rather recently (NAKAMURA, 1978; HAMA, 1978; KAWAKAMI & SASAKI, 1978; NIEUWKOOP, 1973; TAKAYA, 1978; TIEDEMANN, 1978; TOIVONEN, 1978). Unless otherwise indicated, the experiments mentioned in this paper concern Urodele embryos.

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

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.

Immunohistochemical detection of specific antigens in stained sections of the lens mounted in balsam

The location of specific proteins in sections through the lens of amphibians (Ranatemporaria,Xenopuslaevis), stained beforehand with azan and mounted in balsam, was studied by the indirect fluorescent antibodies method. Lenses from normal animals and lenses induced in vitro in the ectoderm of the amphibian gastrula were analyzed. To remove balsam, the lens sections were washed successively in xylol, ethanol, and physiological saline (pH 7.1). They were then treated in accordance with the general principles of immunohistochemical analysis. It is shown that specific antigens can be detected by this method in sections through the lens kept in balsam for 1 year. In sections kept for longer periods (2–3 years) the immunofluorescence test for lens proteins was negative.

NEURAL COMPETENCE AND CELL LINEAGE OF GASTRULA ECTODERM OF NEWT EMBRYO.

The change in the capacity to form neural structures was quantitatively analyzed in both intact and isolated ectoderms of Cynops pyrrhogaster gastrula. The frequency of explants with induced neural structures abruptly decreases between stage 12c and stage 13b in intact ectoderm, and between 12 hr and 18 hr preculture in isolated ectoderm. The quantitative analysis also made clear that the size of the cell population of induced neural structures was gradually reduced with the aging of the ectoderm. The authors simultaneously examined the cell proliferation of early gastrula ectoderm and confirmed that all ectodermal cells divided at least once within 18 hr at 23°C, after which the neural competence of the ectoderm completely disappeared. The relationships between neural competence and cell lineage (cell generation) of the ectoderm are discussed in the light of these findings.

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.

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.

Formation of mesodermal pattern by secondary inducing interactions.

A highly purified vegetalizing factor induces endoderm preferentially in amphibian gastrula ectoderm. After combination of this factor with less pure fractions, a high percentage of trunks and tails with notochord and somites are induced. The induction of these mesodermal tissues depends on secondary factors which may act on plasma membrane receptors of the target cells. The secondary factors are probably proteins as they are inactivated by trypsin or cellulose-bound proteinase K. They are not inactivated by thioglycolic acid.The implication of these findings for tissue determination and differentiation in normal development in relation to the anlageplan for endoderm and mesodermal tissues is discussed.

Studies on the formation and state of determination of the trunk organizer in the newt,Cynops pyrrhogaster.

The exact localization of the presumptive trunk organizer was determined by means of vital staining at the initiation of gastrulation (0 h embryo) and subsequently in 6, 9, 12 and 24 h embryos.The progressive changes in the self-differentiation and inductive capacity of the trunk organizer were studied in isolation cultures (sitting drop) and in sandwich cultures with competent gastrula ectoderm. In the 0 and 6 h embryo cultures the excised trunk organizer predominantly formed atypical ectoderm. A dramatic change in differentiation and inductive capacity occurred in the 9 h embryo. The positive cases - 83% of the isolation and 50% of the sandwich cultures - mainly formed notochord and somites, accompanied by spinal cord and hindbrain in the sandwich cultures. Although no further change in self-differentiation occurred from that time onwards, a gradual increase in inductive capacity was recognized.

Artificial neural crest formation in amphibia.

Early gastrula ectoderm, combined with a denatured neuralizing inducer, was transplanted (exchanged) between different species of urodeles.

Amino acid transport and protein synthesis in induced ectoderm of amphibian embryos.

Isolated gastrula ectoderm ofTriturus alpestris orAmbystoma mexicanum was induced by the vegetalizing factor. Protein synthesis in the induced and uninduced control explants was measured by double labelling with3H-and14C-amino acids after different periods of cultivation. Slight differences were observed in the pattern of nuclear proteins after 12 h of cultivation and in the pattern of cytoplasmic proteins after 48 h of cultivation.The uptake of leucine started to increase in induced explants after 48 h of cultivation and after 96 h was about 50 times greater than in uninduced control explants. The uptake is reduced under partially anaerobic conditions. Ouabain inhibits the uptake by about 50%.

Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass.

Early amphibian gastrula ectoderm (Triturus alpestris) has been treated with vegetalizing factor. While normal sandwiches (animal caps of two eggs) differentiated mainly into endoderm derived tissues, giant-sandwiches (a combination of 8 animal caps) formed mesodermal and neural tissues in addition. The results support the interpretation that ectoderm will differentiate into endoderm derived tissues when all or nearly all cells are induced (presumably depending on certain threshold concentrations of the inducer). This is the case in the normal sandwich after treatment with high concentrations of vegetalizing factor for 24 h. However, in a giantsandwich it must be assumed that only the cells in the vicinity of the inducer will be triggered to differentiate into endoderm derived tissues. Mesodermal structures will be formed by secondary interactions between the induced ectoderm (endoderm) and non induced ectodermal cells. The induction of neural structures could be explained as a further interaction between mesodermalized and non induced ectodermal cells. This chain of events is compared with the steps of determination in normogenesis.

Types of Neural Tissues Induced through the Presumptive Notochord of Newt Embryo

Neural induction through the presumptive notochord was tested by means of the sandwich method. The result disclosed that the notochord was a potent inducer of neural tissues not only in the ectoderm of gastrula but also in the ventral ectoderm of neurula and early tail-bud embryos. Structures formed by the induced neural tissue varied greatly. They can be classified into three types. (1) Tubular: the neural tissues induced in explants containing abundant mesenchymes always formed long tubular structures. The shapes of these neural tubes showed considerable variation; moreover, they were atypical and none formed the regular structure of the spinal cord. This type was most frequent, being found in about 50% of the explants. (2) Inverted: this type was produced when the explant contained mesenchymal component. Consequently, the epithelium of explants was missing. Nevertheless, a considerable mass of neural tissue was always induced. It was noticed that the induced neural tissues were invariably inside out; this type was found in about 30% of the explants.(3) Archencephalic: this was the only type to form the regular structure, i.e., the archencephalon. Formation of the archencephalon was limited solely to those explants containing only a few mesenchymes; this type was found in about 20% of the cases. As described above, it was found that the neural tissues induced by the same inducer of the notochord were not uniform but varied in type. Further it was shown that the types of neural tissue differed according to different quantities of the surrounding mesenchyme. Based on these facts, it is to be concluded that it is not the inducer of notochord, but the surrounding mesenchyme that is of primary importance for the determination of the types of neural tissue.

Influence of cyclic nucleotides on amphibian ectoderm.

Isolated amphibian (Triturus alpestris) gastrula ectoderm was treated with cyclic nucleotides for 24 h and cultured up to 12 days. Explants treated with$cyclic N6-Monobutyryl-adenosine-3'∶5'-monophosphate, cyclic Dibutyryladenosine-3'∶5'-monophosphate and cyclic Dibutyrylguanosine-3'∶5'-monophosphate in a concentration of 10-3 and 10-5 M did not differentiate into mesoderm- or endoderm-derived tissues. The number of explants with small neural and neuroid structures did not exceed the percentage found in the control series. Inductions could also not be obtained when ectoderm was dissociated prior to the treatment with cyclic nucleotides, or when theophylline (which inhibits phosphodiesterase) was added to the culture medium. The results are discussed with regard to the possible mode of action of the vegetalizing factor.

DIFFERENCE IN INDUCTIVE EFFECT OF LIVER TISSUES WITH AND WITHOUT PERISINUSOIDAL BASEMENT MEMBRANE.

Differential inductive capacities among liver tissues of several animals were examined by anticipating the correlation between the capacity and the completness of perisinusoidal basement membrane. The reacting tissue was competent ectoderm of gastrula of Triturus pyrrhogaster, and the inductive effects of livers on the ectoderm were tested by explantation method. The inductive effect of livers being devoid of the membrane (chick and guinea pig) was neural and the tissues having the dense well-developed membrane (reptiles) produced an assembly of neural and meso-dermal tissues, such as notochord and somite or muscle. The livers with the membrane being of intermediate grade of development (calf, Triturus and mouse) induce mesodermal tissues, but not frequently, together with neural tissue or alone. The liver tissue was more active in mesodermal induction in proportion to the completeness of the perisinusoidal basement membrane. On the basis of these data the difference in inductive capacity among liver tissues from different kinds of animals were discussed.

Interaction of induced and uninduced cells during primary induction.

UsingTriturus pyrrhogaster embryos, the effects of uninduced cells on the differentiation of induced cells were investigated. The inducing stimulus was given to the presumptive ectoderm of early gastrulae by treatment with protein sooution from guinea pig bone-marrow. Mesodermal induction was evoked in the ectodermal explants. After the treatment, some of the ectodermal explants were cut into pieces 1/8 of their original size and combined with untreated presumptive ectoderm. Mesodermal tissues were differentiated in the combined explants too, but the mesodermal tissues evoked in these combined ectodermal explants were different in their regional characters from these in uncombined explants; dorsal structures, such as notochrod and muscle, were observed predominatly in the latter, whereas the dominant structures observed in the former were ventral ones, such as mesothelium and mesenchyme. The shifting of the regional characters in the combined explants was regarded as the result of an unknown effect from the uninduced cells.

Inducing activity of fractionated microsomal material from theXenopus laevis gastrula stage.

The postmitochondrial supernatant fromXenopus gastrulae has been fractionated on sucrose gradients. Part of the microsomal material was treated with EDTA, which dissociates most of the polysomal and monosomal material into ribosomal subunits. In addition, a series of pooled fractions from the EDTA treated gradients has been applied to discontinuous gradients in more concentrated sucrose to separate membranous material from the remaining microsomal components.Pooled fractions from all gradients have been tested for inductive activity on amphibian gastrula ectoderm. The spinocaudal (trunk and tail) inducing activity was to some extent eneriched in the membrane fractions.

Transfllter Studies on Neural Induction in the Newt

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 mum were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.