Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

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.     

Early neural crest induction requires an initial inhibition of Wnt signals

Neural crest (NC) induction is a long process that continues through gastrula and neurula stages. In order to reveal additional stages of NC induction we performed a series of explants where different known inducing tissues were taken along with the prospective NC. Interestingly the dorso-lateral marginal zone (DLMZ) is only able to promote the expression of a subset of neural plate border (NPB) makers without the presence of specific NC markers. We then analysed the temporal requirement for BMP and Wnt signals for the NPB genes Hairy2a and Dlx5, compared to the expression of neural plate (NP) and NC genes. Although the NP is sensitive to BMP levels at early gastrula stages, Hairy2a/Dlx5 expression is unaffected. Later, the NP becomes insensitive to BMP levels at late gastrulation when NC markers require an inhibition. The NP requires an inhibition of Wnt signals prior to gastrulation, but becomes insensitive during early gastrula stages when Hairy2a/Dlx5 requires an inhibition of Wnt signalling. An increase in Wnt signalling is then important for the switch from NPB to NC at late gastrula stages. In addition to revealing an additional distinct signalling event in NC induction, this work emphasizes the importance of integrating both timing and levels of signalling activity during the patterning of complex tissues such as the vertebrate ectoderm.     

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.     

Hight performance gel permeation chromatography of proteins

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 glycidoxy-propyltrimethoxysilane 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 of the cell surface charge of amphibian ectoderm after induction

Summary Isolated ectoderm of early gastrula stages ofTriturus alpestris was treated with vegetalizing factor for 24 h employing the sandwich method (induced ectoderm). Controls were incubated for the same period with -globulin which has no inducing activity. Explants of both series were labelled with cationized ferritin, which binds to negatively charged groups at physiological pH. In non-induced ectoderm, ferritin particles can be found as a thin layer all over the plasma membranes. In induced ectoderm the total amount of ferritin bound to the plasma membrane is much lower than in non-induced ectoderm. Ferritin is located in restricted areas only. In contrast to the controls, other membrane areas are free of ferritin particles. The correlation between these results and the change of cell affinity after induction with vegetalizing factor is discussed.     

Tissue-specific changes in nuclear RNA content during early development of Triturus vulgaris

During early development of Triturus vulgaris, as a measure for nuclear activity in neuroectoderm, mesoderm and endoderm, nuclear RNA content was determined by cytochemical methods. In the first stages of gastrulation, that is to say during the early phase of neural induction, the RNA content of the inducing system is considerably higher than in the reacting system. Then, with a phase-shift of about 10 h, the RNA content of the neuroectoderm increases quickly also. In the following stages the nuclear RNA content of both regions is reduced. A second continuous increase in the RNA amount coincides with the formation of the neural tube. In the mesoderm, enhancement of RNA content correlates with cytodifferentiation of the chorda. In all stages the RNA content of the endoderm is higher than in the other tissues and it becomes successively diminished from the early gastrula to the tailbud stage.     

An embryonic inducing factor: isolation by high performance liquid chromatography and chemical properties

A protein (vegetalizing factor) which induces amphibian gastrula ectoderm to tissues which in normal development are derived from endoderm and mesoderm has been isolated from chicken embryo trunks by a combination of size exclusion and reversed phase HPLC. An amount of 0.5 ng factor per gastrula evokes inductions in 80-100% of the cases. The protein (apparent Mr approximately 13 000) is split by NaBH4 to polypeptide chains of about half the size under conditions in which disulfide bridges are reduced. The biological activity is lost.     

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.     

Changes of lectin-binding sites on the embryonic muscle cell surface in the developing ascidian, Halocynthia aurantium

Lectin-binding sites on the muscle cell surface of an ascidian embryo were studied using the ferritin labeling technique. The embryos at 4-cell, gastrula, late tail-bud, and larval stages were dissociated in the Ca²⁺- and Mg²⁺-free solution with or without collagenase. Dissociated cells and fragments were prefixed, reacted with ferritin-lectin conjugates and processed for electron microscopy. Lectins used were concanavalin A (ConA) and Ricinus communis agglutinin. Ferritin particles showing lectin-binding sites were found singly or in the form of clusters on the cell surface exposed directly to the conjugates. Most of the particles of both conjugates were distributed singly and sparsely on entire surface areas of the 4-cell stage cells, whereas they were rich in population and tended to form clusters when embryos reached the gastrula stage. At the succeeding stages tested, tagged ferritin, which was single or clustering particles, was less in number as compared with those at the former stage; on the surface facing neighboring muscle cells, in particular, the ferritin particles were much fewer than those in areas of notochordal and epithelial sides. It is suggested that the embryonic muscle cells of the ascidian show stage-specific changes of cell surface carbohydrates. They have high reactivity to both lectins around the gastrula stage and bring out the regional difference of both lectin-binding sites in the tail-bud stage, namely during the period of histogenesis.     

Tissue-specific changes in nuclear RNA content during early development of Triturus vulgaris

Summary During early development of Triturus vulgaris, as a measure for nuclear activity in neuroectoderm, mesoderm and endoderm, nuclear RNA content was determined by cytochemical methods. In the first stages of gastrulation, that is to say during the early phase of neural induction, the RNA content of the inducing system is considerably higher than in the reacting system. Then, with a phase-shift of about 10 h, the RNA content of the neuroectoderm increases quickly also. In the following stages the nuclear RNA content of both regions is reduced. A second continuous increase in the RNA amount coincides with the formation of the neural tube. In the mesoderm, enhancement of RNA content correlates with cytodifferentiation of the chorda. In all stages the RNA content of the endoderm is higher than in the other tissues and it becomes successively diminished from the early gastrula to the tailbud stage.     

Solubilization and molecular weight determination of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias.

The membrane-bound (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) system was treated with the nonionic detergent octaethylene-glycoldodecyl ether, yielding a transparent supernatant after centrifugation. The supernatant was highly active with both ATPase and p-nitrophenylphosphatase, with initial specific activities of 2300 mumol Pi released . mg-1 protein. h-1 and 350 mumol p-nitrophenol released.mg-1 protein.h-1, respectively. The supernatant was purified to 95--100%, with respect to the 96 000 dalton and the 56 000 dalton peptides. The solubilized enzyme was gel filtered in Sepharose 4B-Cl and displayed 2 peaks, both with catalytic activity. The low molecular weight particles eluted at Kav = 0.54, corresponding to a molecular weight of approximately 500 000 daltons and the particles had a specific activity of 2100 mumol Pi.mg-1 protein.h-1. Both peaks contained phospholipid with 60 mol phospholipid bound per 300 000 g protein. The low molecular weight particles had a molecular weight of 276 000 as determined by sedimentation equilibrium analysis.     

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification.

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.     

In vitro methylation of histones in sea urchin nuclei during early embryogenesis

Nuclei isolated from sea urchin embryos incubated in vitro in the presence of S-adenosyl-[methyl-3H]methionine, methylate their own basic proteins. The protein methylase activity varies during the embryonic development with two peaks of activity at mesenchymal blastula and at young gastrula. Histones H3 and H4 are the main substrates of the reaction. The extent of methylation of the two histones depends on the S-adenosylmethionine concentration. At low S-adenosylmethionine concentrations, the in vitro methyl-accepting ability of H3 is 10-times that of H4, while at high concentrations it is 3-times that of H4. This finding is clearly evident in the equilibrium saturation experiments with blastula and gastrula nuclei, which both show two distinct Km values for S-adenosylmethionine. The major and perhaps only product of methylation is epsilon-N-methyl-lysine. Enzyme activity is clearly correlated with specific embryonic stages, while no correlation is apparent between enzyme activity and the amount of DNA in the embryos.     

Isolation and effects of inducing factors secreted by the lens epithelium cells

Our previous studies showed that, unlike tissue extracts, the cells of living organs secrete substances capable of inducing the same organ rudiments in the early gastrula ectoderm (EGE). In this work, the molecular nature of these substances was studied. The porcine lens epithelium was chosen for the initial analysis. When cultivated, this epithelium secreted a mixture of proteins, which were separated by gel-filtration. Both the total protein mixture and its individual fractions were tested for their inducing capacity using the early gastrula ectoderm of Rana temporaria. Unexpected results were obtained, which indicated that (a) the mixture of native proteins secreted by lens epithelium has a selective inducing capacity differing from those of individual fractions isolated from this mixture and (b) each fraction has a specific effect, but all of them cause the induction of neural tissue or sensory organs. These results (obtained for the first time) suggest that the inducing capacity of individual protein fractions is wider than that of the total protein mixture secreted by lens epithelium. This fact raises a question concerning the relationships between the mechanisms underlying the corresponding inducing effects.     

The effect of temperature on the synthesis and assembly of proticine 3 particles by Proteus mirabilis

Proteus mirabilis CW977 produced high yields of the bacteriocin proticine 3 upon mitomycin C induction of cultures growing at 30 degrees C. The proticine was purified and found to have a relative density of 1.299 and to be composed of 10 proteins assembled into structures resembling contractile phage tails. When induction was performed at 41 degrees C neither proticine particles nor proticine activity was detected, although the growth rate of cells and degree of lysis were indistinguishable from that at 30 degrees C. Failure in proticine production was due to a 41 degrees C sensitive stage occurring between 60 and 90 min after the addition of mitomycin C. During this period at 30 degrees C, two proteins of mol. wt 58 000 and 41 000 were formed. These proteins were associated with events leading to the formation of proticine particles with biological activity. When the production of both proteins was prevented either by chloramphenicol or as a result of mutation or through sampling before they were formed, no proticine particles were found nor proticine activity detected. The synthesis of both proteins was also inhibited at 41 degrees C. Co-electrophoresis of the labelled proteins with unlabelled purified proticine confirmed that the protein of mol. wt 58 000 was a proticine structural protein. The protein of mol. wt 41 000 was not a structural component of proticine and its role, if any, in proticine 3 production is possibly that of an assembly protein.     

Cell Wall Lysin as a Component of the Bacteriophage ?6 Virion

Cell wall lytic activity was found in particles of the lipid-containing bacteriophage ø6. The activity can be extracted from the virion with Triton X-100 in the presence of salt. This treatment removes the membrane-like envelope of the virion which includes five proteins. The lysin requires detergent for in vitro activity. Virus particles formed in nonsuppressor cells by several classes of ø6 nonsense mutants contained the lysin activity; however, particles formed by a mutant (unable to make proteins P5 and P11) had very low activity; high activity was produced when particles were formed in a suppressor host. A study of the time course of the appearance of the lysin during infection showed that it appeared and increased in cells infected with wild-type virus and in suppressor cells infected with a mutant of class 511, but it did not increase in nonsuppressor cells infected with the class 511 mutant. It is concluded that protein P5 is a component of the lysin and that the role of its activity is in both early and late stages of infection. In particular, the lysin may be necessary for the passage of the infecting core of the virion through the cell wall of the bacterium, as well as in the final lysis necessary for the liberation of progeny phage. A mutant of the virus that produces a larger-than-normal protein P10 does not induce normal lysin activity in host Pseudomonas phaseolicola HB10Y, although it does in strain ERA Pseudomonas pseudoalcaligenes. This indicates that protein P5 is probably not sufficient for lysin activity, but the nature of the interaction between P5 and P10 is unknown.     

Curie-point pyrolysis, gas-liquid chromatography of xylan

Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.