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

Summary 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. The experiments suggest that a masked neuralizing factor, which is already present in the ectoderm, is in part activated and exported from the inducing neural plate cells.     

Neural-inducing activity of newly mesodermalized ectoderm

Summary The neural-inducing activity of artificially mesodermalized ectoderm was examined. The competent ectoderm of earlyCynops gastrula was mesodermalized by being placed in contact withCarassius swimbladder. The mesodermalized ectoderm was combined with ectoderm isolated from various developmental stages of a gastrula. Neural differentiation were observed in half the combinants, even in 18 h ectoderm, which is considered to have lost its neural competence within 6 h. This indicates that mesodermalized ectoderm is capable of inducing neural tissues at the very time it comes into contact with 18 h ectoderm. From the present study, the neural-inducing activity of mesodermalized cells may possibly be closely connected to the early process of their mesodermalization.     

Inducing activity of subcellular fractions from amphibian embryos

Summary 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. RNP particles isolated from hatched embryos had a lower inducing activity. The neuralizing factor can be extracted from the small vesicles with pyrophosphate buffer at pH 8.6, but it is not solubilized with a non-ionic detergent (Triton X 100). The high-speed supernatant from the gastrula homogenate contains soluble neuralizing factor, whereas the supernatant from egg homogenate has a low inducing activity. The plasma membrane fraction (isolated from gastrulae) also has only a low inducing activity. The possible significance of the subcellular distribution of neuralizing factors for the transmission of neuralizing inducer from the mesoderm to competent gastrula ectoderm and the processing of signals which are generated on the plasma membrane of induced cells is discussed.     

Covalent coupling of neuralizing factors from Xenopus to Sepharose beads: no decrease of inducing activity

Two neural inducing factors extracted from Xenopus gastrulae, a basic protein from ribonucleoprotein particles and an acidic protein from the high speed supernatant were covalently bound to CNBr-Sepharose or cross-linked CNBr-Sepharose particles. The protein-Sepharose complexes cannot be taken up by the competent ectoderm cells, but both factors remain fully active. The inducing activity is not due to a release of the bound factors. The experiments suggest that both neural inducing factors act on the cell surface of the competent ectoderm cells.     

Neural-inducing activity and glycoprotein synthesis of newly mesodermalized ectoderm in the newtCynops (Amphibia)

Summary An artificially mesodermalized ectoderm (mE) shows the same properties as the organizer: chordamesoderm formation and neural induction. The neural-inducing activity of the mE was inhibited by treatment with protein synthesis inhibitors (cycloheximide and puromycin) and a specific inhibitor of protein glycosylation (tunicamycin). These antibiotics also inhibited chordamesoderm differentiaton, especiallly that of notochord. Newly synthesized proteins of the mE were compared with those of presumptive ectoderm (pE) using two-dimensional PAGE. There were differences in relative amounts of many protein spots. These results suggest that neural-inducing activity is related to glycoproteins synthesized during the early phase of mesodermalization.     

Neural induction in embryos

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g ), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

Partial characterization of neural-inducing factors from Xenopus gastrulae Evidence for a larger protein complex containing the factor

Summary High (Mr 90–110 kDa) and low (Mr 15–30 kDa) molecular weight forms of neural-inducing factors have been found in the supernatant of Xenopus gastrula homogenate. The factors, which are protein in nature, have been partially purified by size exclusion high-performance liquid chromatography (HPLC) and sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The factor of smaller size, which could be derived from a precursor, is associated with other proteins in a larger complex. The neural-inducing factors are not irreversibly inactivated after chemical deglycosylation with trifluoromethansulfonic acid. The neural-inducing protein which is found in ribonucleoprotein (RNP)-particles was partially purified by hydrophobic chromatography. Possible relationships of the factors in different subcellular fractions and their physiological significance are discussed. Offprint requests to: H. Tiedemann     

Homoiogenetic Neural Inducing Activity of the Presumptive Neural Plate of Xenopus Laevis

Heteroplastic combinations were made between Xenopus laevis presumptive neural plate and competent ectoderm of Xenopus borealis . Primarily induced presumptive neural plate cells ( Xenopus laevis ) can easily be distinguished from Xenopus borealis cells by specific quinacrine fluorescence of the nuclei. It was clearly shown that presumptive neural plate, which has primarily been induced by the underlying chordamesoderm exerts homoiogenetic inducing activity on competent ectoderm. The inducing activity is increased in pieces of presumptive neural plates, when the superficial layer has been removed from the adjacent deep layers. The enhancement can be explained by the fact that the removal of the superficial layer acting as barrier allows the inducing stimulus to be easily propagated from the apical (distal) side of the deep layers of the presumptive neural plate.     

Changes in the interior surface of newly mesodermalized ectoderm and its contact activity with competent ectoderm in the newtCynops (Amphibia)

Summary The presumptive ectoderm (pE) ofCynops gastrulae was artificially mesodermalized by contact with teleost swimbladder. The newly mesodermalized ectoderm (mE) acquired the capacity for neural induction (Suzuki et al. 1986a). SEM observations revealed that the mE cells altered their cellular profiles immediately after mesodermalization. The characteristics of the cell surface and the cell architecture became similar to those of invaginated mesoderm cells. There were distinct differences in the cellular contact between mE—pE and pE—pE combinations. The mE-pE combinations kept close contact at their interior surfaces, while the pE—pE combinations did not keep contact. Both TEM and SEM observations also indicated that there were tight contacts between mE and pE cells. These findings suggest that neural-inducing activity of the newly mesodermalized ectoderm cells is coupled with acquisition of cellular affinity toward the interior surface of competent ectoderm cells, and probably requires close cell contacts.     

MITOTIC ACTIVITY AND CELL PROLIFERATION IN PRIMARY INDUCTION OF NEWT EMBRYO

Mitotic activity and cell proliferation of newt ( Triturus pyrrhogaster ) embryo were examined with special reference to primary induction.
Mitotic activity of gastrula ectoderm gradually decreases during gastrulation. The ectoderm, which is isolated from mid-gastrula (stage 12b) and cultured in vitro , also shows gradual decrease in mitotic activity during cultivation and the mitotic activity steeply decreases after 48 hr.
The ectoderm cultured with heterologous inductor (GPL-extract) shows a temporal suppression in mitotic activity. The ectoderm of the whole gastrula also shows a regional suppression where it is in contact with the chorda-mesoderm.
The number of the ectodermal cells increases about 2 times after 24 hr culture and to more than 3 times after 48 hr culture. Accordingly it is certain that the majority of the ectodermal cells divides at least one time in the course of 48 hr.
Histological examination of the ectoderm cultured together with the inductor reveals that differentiation of undifferentiated ectoderm to neural tissues is accomplished at least within 48 hr after cultivation with the inductor.
The present examination shows the possibility that the mitotic activity of the ectoderm may be temporarily suppressed by the inductor and that it then decreases along with neural cell differentiation after recovery of the activity.
The results also suggest that the determination of undifferentiated ectoderm to neural tissues occurs before the second cell division after the contact with the inductor and the events occurring during the first cell cycle after activating by the inducing stimulus are critical for the primary induction.     

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.     

A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus.

The process by which the vertebrate central nervous system acquires its regional properties remains a central problem in developmental biology. It is generally argued that at early gastrula stages the dorsal mesoderm possesses precise anterior-posterior positional information, which is subsequently imparted to the overlying ectoderm. However, using regionally specific gene probes to monitor regional responses in Xenopus embryos, we find that anterior-posterior properties are not fixed until early neurula stages. During gastrulation the regional inducing capacities of the dorsal mesoderm as well as the regional responses of the presumptive neural ectoderm are activated along the entire anterior-posterior axis when these properties are assayed in recombinant and explant experiments, respectively. Restriction of regional inducing capacity in the mesoderm and responsiveness in the neural ectoderm occur only at neural plate stages.     

Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system

In amphibians and other vertebrates, neural development is induced in the ectoderm by signals coming from the dorsal mesoderm during gastrulation. Classical embryological results indicated that these signals follow a “vertical” path, from the involuted dorsal mesoderm to the overlying ectoderm. Recent work with the frog Xenopus laevis, however, has revealed the existence of “planar” neural-inducing signals, which pass within the continuous sheet or plane of tissue formed by the dorsal mesoderm and presumptive neurectoderm. Much of this work has made use of Keller explants, in which dorsal mesoderm and ectoderm are cultured in a planar configuration with contact along only a single edge, and vertical contact is prevented. Planar signals can induce the full anteroposterior (A-P) extent of neural pattern, as evidenced in Keller explants by the expression of genes that mark specific positions along the A-P axis. In this review, classical and modern molecular work on vertical and planar inductionwill be discussed. This will be followed by a discussion of various models for vertical induction and planar induction. It has been proposed that the A-P pattern in the nervous system is derived from a parallel pattern of inducers in the dorsal mesoderm which is “imprinted” vertically onto the overlying ectoderm. Since it is now known that planar signals can also induce A-P neural pattern, this kind of model must be reassessed. The study of planar induction of A-P pattern in Xenopus embryos provides a simple, manipulable, two-dimensional system in which to investigate pattern formation. © 1993 John Wiley & Sons, Inc.     

A comparison of the kinetic properties of mouse brain glutamate decarboxylase activities in the mitochondrial and supernatant fractions

Several properties of the glutamate decarboxylase activities in the high-speed supernatant and water-washed mitochondrial fractions were compared. No significant differences in the values of Km for glutamate, the inhibitory effects of chloride ion, the proportion of pyridoxal-P independent activity remaining after exhaustive dialysis, the effects of added pyridoxal-P or the inhibitory effects of ATP were observed between the two fractions. The water-washed mitochondrial fraction was found to contain about 4 percent of the initial activity in the homogenate. Aside from the fact the small amount of glutamate decarboxylase found in the mitochondrial fraction cannot be released from the particles by washing or other mild treatments, there are presently no well documented differences between the glutamate decarboxylases found in the mitochrondrial and supernatant fractions.     

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.     

Glycoproteins responsive to the neural-inducing effect of Concanavalin A in Cynops presumptive ectoderm

To examine the possible occurrence of receptors in the ectodermal cell surface which apparently mediates the neural-inducing stimulus, a further experiment by using Con A was done in combination with the enzyme treatments. The presumptive ectoderm explants of Cynops gastrula were first treated with neuraminidase to remove sialic acid. Prior to the Con A treatment, the explants were treated with almond glycopeptidase, which cleaves the asparagine linkage between protein and oligosaccharide in glycoprotein and releases the oligosaccharide moiety intact containing mannose residue from the substrate. No neural induction occurred. When the explants were not treated with almond glycopeptidase, the neural induction frequency was found to be the same as that of the explants treated with only Con A. Biochemical analyses showed that when the fixed ectoderm explants were treated with almond glycopeptidase, several oligosaccharides were released and then fractionated by means of Bio-Gel P-4 filtration. Based on the strict specificity of almond glycopeptidase, these oligosaccharides are unmistakably asparagine-linked oligasaccharides with mannose residues. We discuss the hypothesis of involvement of glycoproteins in the first step of molecular events in the neural induction mechanism.