Mesoderm-inducing factors: a small class of molecules

Mesoderm-inducing factors (MIF's) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs-the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other. The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13,000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6.7 (WEHI) and 7.7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose. All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used. Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiations factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.     

Intracellular signalling pathways involved in mesoderm induction by FGF.

We have examined the possible role of two signal transducing mechanisms, tyrosine phosphorylation and activation of protein kinase C (PKC), during fibroblast growth factor (FGF)-induced mesoderm induction in Xenopus. Tyrosine phosphorylation was examined through the use of a monoclonal anti-phosphotyrosine antibody. This antibody was shown to recognize the FGF receptor crosslinked to radioiodinated FGF. We also studied the response of Xenopus ectodermal explants to sodium orthovanadate, a compound that has been shown to elevate intracellular phosphotyrosine levels. Thirty percent of explants cultured in 100 microM vanadate were induced. In addition, vanadate synergized with FGF to give inductions that were more dorsal in nature than either vanadate or FGF alone. The role of PKC was evaluated by measuring PKC activity during mesoderm induction by FGF and by examining the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on explants. TPA did not induce mesoderm, however, activation of PKC was detected in FGF-treated explants. Therefore, activation of the PKC pathway alone is not sufficient for mesoderm induction. Simultaneous treatment with TPA and FGF resulted in a significant inhibition of mesoderm induction by FGF, suggesting that activation of PKC could be part of a negative feedback mechanism. In contrast, TPA had no effect on induction by activin A.     

Mechanism of anteroposterior axis specification in vertebrates. Lessons from the amphibians.

Interest in the problem of anteroposterior specification has quickened because of our near understanding of the mechanism in Drosophila and because of the homology of Antennapedia-like homeobox gene expression patterns in Drosophila and vertebrates. But vertebrates differ from Drosophila because of morphogenetic movements and interactions between tissue layers, both intimately associated with anteroposterior specification. The purpose of this article is to review classical findings and to enquire how far these have been confirmed, refuted or extended by modern work. The "pre-molecular" work suggests that there are several steps to the process: (i) Formation of anteroposterior pattern in mesoderm during gastrulation with posterior dominance. (ii) Regional specific induction of ectoderm to form neural plate. (iii) Reciprocal interactions from neural plate to mesoderm. (iv) Interactions within neural plate with posterior dominance. Unfortunately, almost all the observable markers are in the CNS rather than in the mesoderm where the initial specification is thought to occur. This has meant that the specification of the mesoderm has been assayed indirectly by transplantation methods such as the Einsteckung. New molecular markers now supplement morphological ones but they are still mainly in the CNS and not the mesoderm. A particular interest attaches to the genes of the Antp-like HOX clusters since these may not only be markers but actual coding factors for anteroposterior levels. We have a new understanding of mesoderm induction based on the discovery of activins and fibroblast growth factors (FGFs) as candidate inducing factors. These factors have later consequences for anteroposterior pattern with activin tending to induce anterior, and FGF posterior structures. Recent work on neural induction has implicated cAMP and protein kinase C (PKC) as elements of the signal transduction pathway and has provided new evidence for the importance of tangential neural induction. The regional specificity of neural induction has been reinvestigated using molecular markers and provides conclusions rather similar to the classical work. Defects in the axial pattern may be produced by retinoic acid but it remains unclear whether its effects are truly coordinate ones or are concentrated in certain regions of high sensitivity. In general the molecular studies have supported and reinforced the "pre-molecular ones". Important questions still remain: (i) How much pattern is there in the mesoderm (how many states?) (ii) How is this pattern generated by the invaginating organizer? (iii) Is there one-to-one transmission of codings to the neural plate? (iv) What is the nature of the interactions within the neural plate? (v) Are the HOX cluster genes really the anteroposterior codings?     

Toxin Production in Clostridium botulinum as Demonstrated by Electron Microscopy

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.     

Evidence suggesting that virulence maps to the P1 region of the coxsackievirus B4 genome.

A chimeric virus containing the P1 region of a virulent variant of coxsackievirus B4 and the P2 and P3 regions of a nonvirulent strain was constructed from cDNA clones. The chimeric virus induced pancreatitis with concurrent hypoglycemia similar to that observed in mice infected with the virulent variant.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.