Determination, induction and pattern formation in early amphibian embryos.

Determination (inducing) factors, the extracellular matrix, signaling pathways, transcription factors and genes interact in pattern formation and neural induction. Genes can either be activated or repressed. The animalvegetal and dorso-ventral polarities are determined in very early developmental stages.

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 μg/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.

Proteoglycans with affinity for the neuralizing factor and the vegetalizing factor (activin A homologue).

Proteoglycans from chicken embryos bind neuralizing and vegetalizing inducing factors. The proteoglycan-factor complexes have no inducing activity. Enzymatic cleavage of the core proteins of the proteoglycans abolishes inhibition of the inducing activity by proteoglycans.

A mesoderm-inducing factor from a Xenopus laevis cell line : Chemical properties and relation to the vegetalizing factor from chicken embryos.

We have compared the chemical properties and biological activities of the mesoderm-inducing factor that is secreted by the Xenopus XTC cell line with the vegetalizing factor from chicken embryos. The inducing activity of the factors was tested in different concentrations on totipotent ectoderm either by implantation into early gastrulae of Triturm alpestris or by application of solutions to isolated ectoderm of early gastrulae of Xenopus laevis. Both factors have similar properties.

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester.

Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis.

The activation of a neuralizing factor in the neural plate is correlated with its homoiogenetic-inducing activity.

Neural plates which are induced in the dorsal ectoderm of Triturus by the underlying mesoderm acquire, in turn, neural-inducing activity. This process is correlated with the appearance of neural-inducing activity in the microsomal fraction of the neural plate homogenate. The high-speed supernatant also acquires inducing activity after neural induction, but to a lesser extent.

Isolation of plasma membranes from Xenopus embryos.

Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers.

Activation of a neuralizing factor in amphibian ectoderm.

Isolated gastrula ectoderm has no neural-inducing activity and does not differentiate into neural tissues. It has, however, a high neural-inducing capacity, but the inducing factors are present in a masked, inactive form. The inducing factors are partially activated by homogenization and by freezing of the homogenate and are fully activated by treatment with ethanol.

Inducing activity of subcellular fractions from amphibian embryos.

The homogenate from unfertilized eggs, gastrulae, neurulae and hatched embryos ofXenopus laevis was fractionated by differential centrifugation and subsequent repeated centrifugation on discontinuous sucrose gradients. A high archencephalic-neural inducing activity was found in RNP particles, which were released from the high-speed ("microsomal") sediment by treatment with EDTA, and in a fraction of heterogeneous small vesicles. The highest archencephalic inducing activity was observed in RNP particles from unfertilized eggs and from gastrulae.

Neural induction in amphibians : Transmission of a neuralizing factor.

The neural plates of very early neurula stages of Triturus alpestris were removed, the material which is released from the extracellular space between mesoderm and neural plate to the medium in which the embryos were dissected was isolated and extracted with phenol. The protein isolated from the phenol layer showed neural inducting activity. Proteoglycans isolated from the aqueous layer did not show such inducing activity.

Biological activity of the vegetalizing factor: Decrease after coupling to polysaccharide matrix and enzymatic recovery of active factor.

The inducing activity of the vegetalizing factor decreases after covalent coupling to CNBr-Sepharose with reduced binding capacity. The residual inducing activity is probably due to the release of a small amount of the factor from Sepharose beads. Covalent coupling to activated CH-Sepharose completely inactivated the vegetalizing factor, whereas the neuralizing factor retained its full activity.

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.

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 withH-andC-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.

Electrophoretic spectra of nuclear proteins from embryos ofXenopus laevis.

The changes in saline-soluble, 0.35 M NaCl-soluble and the residual fraction of nuclear proteins during early development ofXenopus were studied by analytical electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The fractions were obtained by consecutive extraction of nuclei from the blastula, neurula and tail-bud stage of development.

Mechanisms of cell differentiation: Affinity of a morphogenetic factor to DNA.

A morphogenetic factor which induces inTriturus gastrula ectoderm tissues which are derived from mesoderm and endoderm has been extracted from chicken and amphibian embryos. The factor which is protein in nature has been obtained from chicken embryos in a highly purified state.The biological activity of the chicken factor is partially inhibited when the factor is combined with chicken DNA or sonicated chicken DNA.

[Action of sulfhydryl compounds on embryonic inducing factors].

Crude extracts from 9 days old chicken embryos containing neuralizing and mesodermalizing inducing factors as well as purified mesodermalizing factor were incubated with thioglycolic acid and with 2-Mercaptoethanol. The fractions were tested by implanting into early gastrulae ofTriturus orAmbystoma. The mesodermalizing factor is inactivated whereas the neuralizing factor does not lose its activity.

[The inducing capability of nucleic acids tested on amphibian ectoderm].

Ribonucleic acids were extracted from chick- andTriturus embryos by the phenol method and tested for their inducing ability:Ribonucleic acids from chick embryos had only a weak inducing capacity compared with that exerted by inducing chick embryo protein fractions. In tissue cultures of undetermined amphibian ectoderm they induced unspecific neural structures, mesenchymal cells and melanophores only to a small extent. But the differentiated part of the tissue was still higher than in control series where the ectoderm was cultivated in Flickinger solution alone.