INDUCTIVE CAPACITIES OF GLOMERULAR BASEMENT MEMBRANES AND DENTINE MATRIX

The inductive capacities of the basement membranes of calf kidney glomeruli and the dentine matrix of the incisors of 23-day rabbit fetuses were examined on the presumptive ectoderm of Triturus gastrulae. The basement membranes caused almost entirely neural induction and the dentine matrix caused mesodermal induction. These findings suggest that intercellular substances play an important role in the inductive effects of heterologous tissues.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

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.     

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

Summary 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.     

Inductive characteristics of proteins secreted by retinal cells

The studies of the development of eye rudiments and formation of adult eye tissues have always been among priorities in developmental biology and then in developmental genetics, which is associated with the peculiarities of the development and structure of the eye. In the late 80s, it was established by the group of developmental factors of the Institute of Gene Biology of RAS that many differentiated tissues are able to produce proteins causing homologous differentiations in polypotent cells of early gastrula ectoderm. The aim of our present study was isolation of proteins secreted by mammalian and fish retinal cells and determination of their inductive properties in early gastrula ectoderm of Xenopus laevis. The sets of proteins secreted by retina induce tissues homologous to the inducer, that is, neural tissue, brain, retina, pigmented epithelium, and also lenses and ear vesicles. The retinal inductive proteins retain their homologous inductive capacity after lyophilization. Biological testing shows that a total mixture of proteins secreted by retinal cells induces in polypotent gastrula ectoderm of X. laevis a narrower spectrum of tissues than the fractions obtained from this mixture. The above-outlined results obtained in thecourse of investigations of inductive peculiarities of retina and its fractions help in the elucidation of questions concerning embryonic induction and factors determining it, as well as questions concerning the maintenance of tissue specifity and regenerative capacity of the tissue studied.     

TEMPORAL RELATIONS BETWEEN EXTENSION OF ARCHENTERON ROOF AND REALIZATION OF NEURAL INDUCTION DURING GASTRULATION OF NEWT EMBRYO

This investigation was performed in order to analyze the basic relationships between the archenteron roof and the overlying ectoderm in primary induction in the Cynopus (Triturus) pyrrhogaster embryo.
The part of the archenteron roof that is active in inducing capacity extends linearly after invagination at the speed of 0.15 mm per hr at 23°C until stage 13b. The period of contact at each position of the presumptive neuro-ectoderm with the active archenteron roof could be estimated by the formula described in the Discussion.
Pieces of the presumptive neuro-ectoderm were isolated from gastrulae at three developmental stages and cultured separately in Holtfreter solution after being divided caudo-cranially into 4 parts. The result showed that some of them were able to differentiate into neural tissues even in the mid-gastrula stage and that the presumptive neuro-ectoderm acquired the capacity to differentiate into neural tissue along a caudocranial axis from the part adjacent to the blastopore during gastrulation.
It could be estimated that 3 hr of contact with the active archenteron roof is sufficient for the presumptive neuro-ectoderm to differentiate into neural tissue.
The present study also showed that the neuralizing capacity of the whole prospective neuro-ectodermal area has already been determined before the end of stage 13, i.e., within less than 14 hr after first contact of the ectoderm with the active archenteron roof at 23°C.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Basic fibroblast growth factor can induce exclusively neural tissue in Triturus ectoderm explants

Ectoderm was isolated from early gastrulae of Triturus alpestris and induced with recombinant basic fibroblast growth factor (b-FGF). Neural tissue differentiated in about 38% of the explants which were induced by 2,5 g/ml FGF. These explants do not contain other tissues, or contain only small amounts of mesenchyme and melanophores which are probably derived from induced neural crest. It is therefore unlikely that these neural tissues are secondarily induced. The other explants contain predominantly blastema tissue, endothelium/ mesothelium, small amounts of skeletal muscle and, rarely, notochord besides neural tissues. The mitotic rate was enhanced in about 20% of the induced explants. Possible mechanisms for the unexpected neural-inducing activity of b-FGF in Triturus ectoderm 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.     

Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration

Immunofluorescence and immunoperoxidase labeling for fibronectin was used to study the early events of cephalic neural crest cell migration in avian embryos. Prior to crest cell appearance, fibronectin was associated with the basement membranes of all tissues. The loose mesenchymal cells were also surrounded by this glycoprotein. The crest cell individualization phase included a transient rounding up and a rapid increase in cell number in a very limited space. Whereas the neural tube basement membrane was not formed dorsally at the site of emergence of crest cells, it was partially fused laterally with the ectoderm basement membrane apparently preventing immediate crest cell emigration. Further increase in cell number occurred concomitantly with their penetration between the two developing basement membranes of the neural tube and the ectoderm. The localization of migrating crest cells is apparently greatly influenced by local interactions between the ectoderm and the neural tube, whose morphogenesis differs considerably at each axial level: at the mesencephalic-rhombencephalic levels, crest cells rapidly reached a cell-free space that was mostly devoid of fibronectin. Further migration occurred laterally in that space while pioneer crest cells became surrounded by fibronectin in their environment. Crest cells progressed as a confluent multicellular layer with an apparent velocity of 70 μm/hr. At the prosencephalic and median rhombencephalic levels, crest cells accumulated between the fibronectin-rich basement membranes of the ectoderm and the neural tube. Pioneer crest cells were arrested at the site of attachment of the ectoderm and the neural tube basement membranes (i.e., optic vesicles and otic placodes). Crest cells resumed their migration when more space became available during the constriction of the optic vesicles and the invagination of the otic placodes.     

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.     

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.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments

Summary The inducing capacity of the vegetal hemisphere of early amphibian blastulae was studied by placing a Nucleopore filter (pore size 0.4 m) between isolated presumptive endoderm and animal (ectodermal) caps. The inducing effect was shown to traverse the Nucleopore membrane. The reacting ectoderm differentiated into mainly ventral mesodermal derivatives. Expiants consisting of five animal caps also formed dorsal mesodermal and neural structures. Those results together with data published elsewhere suggest that, in addition to a vegetalizing factor, different mesodermal factors must be taken into consideration for the induction of either the ventral or the dorsal mesodermal derivatives. The neural structures are thought to be induced by the primarily induced dorsal mesodermal tissue. Electron microscopic (TEM) examination did not reveal any cell processes in the pores of the filter. The results indicate that transmissible factors rather than signals via cytoplasmic contacts or gap junctions are responsible for the mesodermal induction of ectodermal cells. The data support the view that in normogenesis the mesoderm is determined by the transfer of inducing factors from vegetal blastomeres to cells of the marginal zone (presumptive mesodermal cells).     

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.     

Cell Contacts Between Chorda-Mesoderm and the Overlaying Neuroectoderm (Presumptive Central Nervous System) During the Period of Primary Embryonic Induction in Amphibians

Using transmission and scanning electron microscopy we were able to show that during primary embryonic induction in amphibians ( Triturus alpestris ) the interspace between the inducing chorda-mesoderm and the reacting ectoderm (presumptive medullary plate) of mid-gastrula stages is traversed by cell projections starting from cells of both tissue layers. In addition intimate membrane contacts between the main bodies of the ectodermal and chorda-mesodermal cells could be observed.
It could be ruled out that cytoplasmic bridges (anastomosis) exist between cells of inducing chorda-mesoderm and reacting ectoderm, which would allow a free transfer of inducing substances without passing through membranes, as Eakin and Lehmann [1] have postulated. The possible role of cell to cell contact for neural induction is emphasized.     

Cell contacts between chorda-mesoderm and the overlaying neuroectoderm (presumptive central nervous system) during the period of primary embryonic induction in amphibians.

Using transmission and scanning electron microscopy we were able to show that during primary embryonic induction in amphibians (Triturus alpestris) the interspace between the inducing chorda-mesoderm and the reacting ectoderm (presumptive medullary plate) of mid-gastrula stages is traversed by cell projections starting from cells of both tissue layers. In addition intimate membrane contacts between the main bodies of the ectodermal and chorda-mesodermal cells could be observed. It could be ruled out that cytoplasmic bridges (anastomosis) exist between cells of inducing chorda-mesoderm and reacting ectoderm, which would allow a free transfer of inducing substances without passing through membranes, as Eakin and Lehmann [1] have postulated. The possible role of cell to cell contact for neural induction is emphasized.     

Effect of concanavalin A and vegetalizing factor on the outer and inner ectoderm layers of early gastrulae of Xenopus laevis after treatment with cytochalasin B

Neural (archencephalic) structures have been evoked in the competent ectoderm (consisting of both ectodermal layers) of Xenopus laevis by treatment with Concanavalin A (Con A), which probably acts on the plasma membrane. The size of the neural structures is increased when the ectoderm is incubated in Cytochalasin B prior to the Con A treatment. The results indicate that Cytochalasin B could have an influence on the binding of Con A to receptors on the plasma membrane. On the other hand, Cytochalasin B seems to have an inhibitory effect on the action of the vegetalizing factor, which could be correlated with the decline of endocytotic processes and internalization. In further series, it could be shown that the isolated superficial ectoderm, in contrast to the inner ectoderm layer, does not react to Con A treatment with the differentiation of neural structures. Studies with FITC-Con A indicate that the marker binds less to the outer ectoderm than to the inner ectoderm layer. However, by xenoplastic combinations of the outer ectoderm layer of X. laevis as reacting tissue and chordamesoderm of Triturus vulgaris as inducer, it could be demonstrated that the superficial layer, which is normogenesis does not come into contact with the inducing chordamesoderm but forms the ependymal part of the brain only, is also able to form archencephalic brain structures under in vitro conditions.     

Transmission Problem in Primary Induction

It has been suggested that during the neuralization step of primary induction in the amphibian embryo the inductive signals are mediated from mesodermal cells to the responding competent neuroepithelium by means other than cell contacts. This idea corroborated by experiments in which the interacting tissues were separated by a Nuclepore filter with pores of 0.05 μm (series 1) or by a dialyzing membrane with pores of only 12,000 daltons (series 2). After 18–22 h exposure to mesoderm followed by 8–10 days' culture in isolation the ectodermal explants were neuralized in both series with about 80% differentiating into archencephalic structures. These results exclude the possibility of cell contact as a mediating mechanism in this step.
In a third series similar experiments were made using a special Nuclepore filter with dense pores of 0.6 μm and the exposure time was prolonged to 24 h. During subsequent culture in isolation the ectoderm was neuralized in every case, except forebrain, the ectoderm also differentiated in 25% to hindbrain and less frequently to spinal cord, myotomes, and in some cases even to notochord. The result is interpreted to mean that during the prolonged exposure the tissues have had time to age to an early neurula stage, and the ectodermal cells, after being neuralized, have had time to form cell contacts by cytoplasmic bridges through the pores resulting in the segregation of the preneuralized ectoderm into more caudal structures than the forebrain.     

The molecular mechanism of neural induction: neural differentiation of Triturus ectoderm exposed to Hepes buffer

Summary Ectoderm was isolated from early gastrula stages of Triturus alpestris and cultured in salt solution buffered with either bicarbonate or Hepes as the principal buffer substance. When bicarbonate was the principal buffer substance or when bicarbonate was omitted, the isolated ectoderm formed atypical epidermis. When Hepes was added as a buffer substance, neural tissue was formed in a high percentage of cases. The differentiation of neural tissue depends on the pH of the Hepes buffer. Hepes in the protonated form, which prevails at lower pH, seems to evoke neural differentiation at a much higher rate. Hepes could either enhance the NA⁺/H⁺ antiport system or it could directly bind to plasma membrane constituents. In both cases conformational changes in the plasma membrane could generate signals which finally lead to neural differentiation.