From presumptive ectoderm to neural cells in an amphibian

As an immediate consequence of neural induction during gastrulation, some neuroectodermal cells acquire the ability to develop a number of specific neuronal and astroglial features, without requiring subsequent chordamesodermal cues. Thus, cholinergic, dopaminergic, noradrenergic, gabaergic, somatostatinergic, enkephalinergic, etc. traits are expressed in cultures of neural plate and neural fold isolated from amphibian late gastrulae immediately after induction and cultured in a defined medium. These results strongly suggest that at the late gastrula stage, the neural precursor population does not yet constitute a homogeneous set of cells. It was of interest to know the origin of this heterogeneity. Is it a direct result of the process of neural induction itself, stochastic phenomena being involved or not at the cellular level, or does it reflect a pre-existing heterogeneity in the presumptive ectoderm? At the early gastrula state, presumptive ectoderm can be neuralized consecutively to its dissociation into single cells. Using this experimental model, we have demonstrated by means of immunological probes that neuralized presumptive ectodermal cells, without any intervention of the chordamesoderm (natural inducing tissue), can develop autonomously into glial and neuronal lineages. These data suggest the existence of diverse predispositions of presumptive ectodermal cells. Competent ectoderm seems to be a heterogeneous structure with cells presenting distinct neural predispositions that can emerge as a consequence of a permissive inductive signal without real specificity (such as a target tissue dissociation). Moreover, such a differentiated neuronal population includes neurons of the GABAergic and enkephalinergic phenotypes but not of the cholinergic, catecholaminergic, somatostatinergic, etc. phenotypes. These data show that the developmental program of ectodermal cells induced without interaction with the chordamesoderm appears restricted compared to the naturally induced ectoderm. Experiments are now under way to analyze such sequential neural events.     

Regional segregation of ConA receptors on dissociated amphibian embryo cells.

Superficial ectoderm cells from early amphibian embryos maintain regional specializations of their cell surfaces, both in intact tissue and as single cells which have been dissociated with EDTA. The cells are adhesive on lateral and basal cell surfaces and non-adhesive on the apical surface. This study presents evidence that these cells display a regional segregation of ConA receptors. On dissociated superficial ectoderm cells, ConA receptors are restricted to lateral and basal surfaces, with the apical surface having few or no receptors. The results are discussed with respect to possible correlations with regional membrane differences in adhesiveness and in regard to the mechanisms which might be involved in maintaining the regional segregation of ConA receptors.     

Comparative study of dieldrin effects on amphibian liver and early embryo phospholipids

1. The effect of Dieldrin on phospholipid content and composition was compared in adult frog liver and in early embryos. 2. Male frogs are more resistant to acute Dieldrin treatment than females. Total phospholipids content increases in the liver of male animals while in females decreases. 3. Early embryo development was not affected morphologically by the addition of 0.02-10.0 mg of Dieldrin/1; however phospholipid content and composition was altered. 4. Adaptive changes in the embryo due to chronically administered Dieldrin is suggested.     

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.     

Gravitational effects on biological systems

The possible effects of the earth's gravitational field on biological systems have been studied from a quantitative point of view, focusing the attention to a very simple system, a solution containing proteins, which biochemists might use in experiments. Gravity has been compared with other forces which are known to influence protein activity, including thermic agitation, weak electrostatic interactions, Van der Waals forces and viscous dissipation. Comparisons have been described in terms of the energy of the interaction per mole, referring to some physically simple cases and substances of biological interest. From this study it is evident that the earth's gravitational energy should be taken into account when considering the chemical behaviour of solutions containing substances that have high molecular weight, such as a typical protein, since its value is comparable to other weak interactions. Moreover, since solutions represent the basis of much more complex biological processes taking place inside cells, the influence of gravity should extend also to cellular biochemical behaviour, especially in presence of altered gravity, both in microgravity (such as on satellites orbiting around the earth), and in macrogravity (such as in a centrifugating biological system).     

Rayleigh instability of the inverted one-cell amphibian embryo

The one-cell amphibian embryo is modeled as a rigid spherical shell containing equal volumes of two immiscible fluids with different densities and viscosities and a surface tension between them. The fluids represent denser yolk in the bottom hemisphere and clearer cytoplasm and the germinal vesicle in the top hemisphere. The unstable equilibrium configuration of the inverted system (the heavier fluid on top) depends on the value of the contact angle. The theoretically calculated normal modes of perturbation and the instability of each mode are in agreement with the results from ComFlo computational fluid dynamic simulations of the same system. The two dominant types of modes of perturbation give rise to axisymmetric and asymmetric sloshing of the cytoplasm of the inverted embryos, respectively. This work quantifies our hypothesis that the axisymmetric mode corresponds to failure of development, and the asymmetric sloshing mode corresponds to development proceeding normally, but with reversed pigmentation, for inverted embryos.     

The neuroanatomy of an amphibian embryo spinal cord

Horseradish peroxidase has been used to stain spinal cord neurons in late embryos of the clawed toad (Xenopus laevis). It has shown clearly the soma, dendrites and axonal projections of spinal sensory, motor and interneurons. On the basis of light microscopy we describe nine differentiated spinal cord neuron classes. These include the Rohon-Beard cells and extramedullary cells which are both primary sensory neurons, one class of motoneurons that innervate the segmental myotomes, two classes of interneurons with decussating axons, three classes of interneurons with ipsilateral axons and a previously undescribed class of ciliated ependymal cells with axons projecting ipsilaterally to the brain. We believe that all differentiated neuron classes are described and that this anatomical account is the most complete for any vertebrate spinal cord.     

Gravitational effects on the response to different stimulatory signals in T cells

The purpose of this paper is to present new data on the in vitro activation of purified T cells with three different mitogenic agents in conditions simulating low-gravity.     

Beneficial effects of embryo extract on cultured rabbit cartilage cells

Growth and the expression of cartilage-like differentiation by adult rabbit chondrocytes is stimulated greatly by the Sephadex G-25 macromolecular fraction of chicken embryo extract (CEEM). A small amount of fetal bovine serum or the macromolecular fraction of fetal bovine serum (FBSM) is also needed for growth. Medium F12 supplemented with CEEM plus FBSM supports moderate clonal growth of the rabbit chondrocytes with excellent expression of differentiated properties. The effects of CEEM on growth and the expression of differentiation have been quantitated by measurement of radioactive sulfate incorporation and total protein synthesis.     

Morphological differentiation of mitochondria in the early amphibian embryo

Three different cell types (epidermal cells, fibroblast-like Ruffini cells and undifferentiated cells) may be observed in cultures of cells isolated from the blastula of Ambystoma mexicanum. Electron micrographs show that the mitochondria in the two kinds of differentiated cells have the orthodox conformation; those in the undifferentiated cells the condensed conformation. Measurements of the area and the form factor demonstrate that the mitochondria in the differentiated cells are considerably larger than those in the undifferentiated cells, and that the three populations of mitochondria differ significantly from each other.     

Inhibition of neural crest cell differentiation by embryo ectodermal extract.

The white mutation in Mexican axolotls has long been thought to be a defect associated with the embryonic extracellular environment, but not with embryonic neural crest cells. Thus it was believed that pigment cells in white axolotls disappear from the skin during early development, not because they are intrinsically defective but because they have no choice but to move into an unfavorable environment. We present evidence to suggest that: (1) white neural crest cells are in fact intrinsically different from dark (wild-type) cells, and (2) an inhibitor is produced in white embryonic ectoderm that actively suppresses the migration, differentiation, and survival of pigment cells in this animal. How these observations fit into the existing body of literature on the white mutant and a model for how the white phenotype might develop are discussed.