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.     

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-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.     

Structural and functional characterization of an anti‐West Nile virus monoclonal antibody and its single‐chain variant produced in glycoengineered plants

Previously, our group engineered a plant‐derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single‐chain variable fragment (scFv) fused to the heavy chain constant domains (C_H) of human IgG (pE16scFv‐C_H). pE16 and pE16scFv‐C_H were expressed and assembled efficiently in Nicotiana benthamiana ?XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ?XF plant‐derived pE16scFv‐C_H (?XFpE16scFv‐C_H) and pE16 (?XFpE16) both displayed a mammalian glycosylation profile. ?XFpE16 and ?XFpE16scFv‐C_H demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (mE16). A single dose of ?XFpE16 or ?XFpE16scFv‐C_H protected mice against WNV‐induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.     

Cell-to-cell contact in primary embryonic induction: effects of lectin on electrical coupling and neural induction

The effects of lectin (concanavalin A; ConA) on the electrical coupling between inducing chorda-mesoderm and reacting ectoderm cells, and the realization of neural induction were investigated. The electrical coupling between cells of the chorda-mesoderm of the late gastrula (stage 13b) and the competent ectoderm or Con-A-treated ectoderm of the early gastrula (stage 12a) was measured. Neural induction was tested with ectoderm explants which had been combined with the inducing chorda-mesoderm for 1, 3 and 6 h. Electrical coupling was observed after 3 h. By 6 h, the coupling ratio had recovered to the same level as that between the homogeneous germ-layer cells. However, the electrical coupling did not recover in the combinant with Con-A-treated ectoderm. This suggests that Con-A disturbs close cell contact between the ectoderm and chorda-mesoderm cells. Neural induction was realized in the ectoderm which was combined with chorda-mesoderm for more than 3 h; this occurred parallel to the recovery of electrical coupling. In contrast, Con-A treatment (50 micrograms/ml) of the competent ectoderm for 30 min prevented neural induction. After 3 h of contact, the neural induction of Con-A-treated ectoderm was only one-third of that of the control ectoderm. The present study suggests that cellular contact between the inducing mesoderm and the ectoderm target cells plays an important role in the realization of neural induction.     

Autolytic activity associated with competent group H streptococci

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.     

Neural Induction by Treatment with Ca2+-free Saline Solution in the Gastrula Ectoderm of Cynops pyrrhogaster and Inhibition of Neural Induction with Fetal Calf Serum

The relation between the inducing activity and the cell-dissociation effect of Ca²⁺-free (or Ca²⁺, Mg²⁺-free) saline solution (CF or CMF) on the early gastrula ectoderm was examined. In the culture medium containing no fetal calf serum (FCS), most ectoderm cells treated with CF or CMF died within a few days and only a few differentiated into epidermal cells. However, when the culture medium contained 2% FCS, ectoderm cells treated with CF or CMF differentiated into neural crest derivatives (NCDs), such as mesenchyme cells, pigment cells, and nerve cells. The frequency of the induction depended only on the duration of CF- or CMF-treatment. FCS alone had no inducing activity on ectoderm cells. On the contrary a high concentration of FCS gave an inhibitory effect on the induction. These results indicate that CF is a neuralizing factor and that CF-treated cells require FCS, not for induction, but for survival and differentiation. With CF, the maximum induction of NCDs required a longer duration than that necessary for complete cell-dissociation. This result suggests that the induction depends on some effects of CF other than cell-dissociation.     

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.
Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof.
The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.     

Electron microscope study of the binding of Con A-gold to superficial and inner ectoderm layers ofXenopus laevis and its relation to the neural-inducing activity of this lectin

Summary Isolated competent amphibian ectoderm differentiates into neural (archencephalic) structures when treated with the plant lectin concanavalin A (Con A). While the inner ectoderm layer ofXenopus laevis forms brain structures after incubation with Con A, the outer ectoderm layer differentiates into ciliated epidermis only. This difference can be correlated with the pattern of Con A bound to the plasma membrane. With gold-labelled Con A it could be shown by transmission electron microscopy (TEM) that the outer ectoderm binds substantially less lectin than the inner layer. Furthermore we observed characteristic differences at the apical and basal surfaces of the cells of the same layer, i.e. on the apical cell surface of the superficial layer almost no Con A-gold could be found. In contrast, we observed a lot of gold particles on the basal cell side of the superficial layer. However, the number on both surfaces (apical and basal side of the cell) of the inner ectoderm layer was essentially higher, which could explain its biological reaction to the Con A stimulus and the differentiation into neural structures. The data presented in this paper indicate that early and late gastrula ectoderm bind similar amounts of Con A and support the view that the decrease in competence is not correlated with a loss of receptors for inducing factors. Furthermore, we describe the binding and the internalization of Con A via receptor-mediated endocytosis and the further fate of the Con A-gold-receptor complex inside the target cell.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

Partial purification of a competence factor from Rhizobium japonicum

A competence factor (CF) from Rhizobium japonicum was partially purified to 43 fold on Sephadex G-100. This CF preparation was sensitive to heat, trypsin and pronase, was resistant to DNase 1, RNase A and lysozyme. It had an approximate mol. wt. of 82,000. Osmotic shock treatment of competent cells revealed that the CF is located in the periplasmic region of the cell.Abbreviations CF competence factor - BSA bovine serum albumin - YM yeast mannitol medium     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

Comparative study of sequential expression of the organizer-related genes in normal Cynops pyrrhogaster embryos and mesodermalized ectoderm

An artificially mesodermalized ectoderm (mE) of early Cynops pyrrhogaster gastrula acquires the organizer property; the mE is able to induce the secondary axis. The expression of organizer-related genes was investigated during the mesodermalizing process of the mE. The expression of C. pyrrhogaster organizer-related genes, such as bra, gsc, lim-1, chd and noggin, were analyzed. Cynops pyrrhogaster shh expression was also investigated. The organizer-related genes were activated by 12 h after the mesoderm-inducing stimulus. It was noted that there was a temporal gap in the expression of each gene. The expression of bra and gsc seemed to be more quickly activated during the mesodermalizing process. While expression of lim-1 and noggin was activated later than that of bra and gsc, lim-1 expression was earlier than chd and noggin expression. Shh expression was activated later than lim-1/noggin. The present study suggests the possibility that the bra/gsc, lim-1, chd, noggin and shh genes are expressed one by one in that order during the mesodermalizing of the presumptive ectoderm. It also indicates that the sequence is not always consistent with that of the whole embryo during normal embryogenesis. The meaning of the discrepancy will be discussed in connection with the cascade of certain genes expressed during the mesodermalizing process.     

The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo

Summary Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating³H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of³H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.     

Extracellular matrix mediates epithelial effects on chondrogenesis in vitro

It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.     

Linkage of the DNA-segment D7S13 (pB79a) with the cystic fibrosis locus

Summary A map distance of 2.9 cM between D7S13 (pB79a) and the cystic fibrosis (CF) locus was obtained from the analysis of 13 informative families with a history of CF. This result is based solely on the HindIII restriction fragment length polymorphism (HindIII-RFLP) at D7S13, since the interpretation of the Msp1-RFLP at this locus was found to be unreliable.     

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.     

DIFFERENTIATION OF PARTIALLY MESODERMALIZED ECTODERM; HOMOIOGENETIC AND HETEROGENETIC INDUCTION BY PRIMARILY INDUCED PART OF THE ECTODERM

Using embryos of the Japanese newt, Cynops pyrrhogaster , homoiogenetic and heterogenetic induction were investigated in the partially mesodermaelzed presumptive ectoderm. Half of the isolated presumptive ectoderm was placed in contact with the swimbladder of the crucian carp, Carasius auratus , for 15 or 60 min, while the other half was stained with Nile blue sulfate at the same time. The distribution of the stained cells in the tissues evoked in the explants was examined after cultivation for 10 days.
Some mesodermal tissues were composed of both stained and unstained cells. This indicates homoiogenetic induction by the primarily induced part of the ectoderm on the other half. The neural and epidermal tissues in the explants were composed of stained cells only, except in one case. We conclude that the neural tissues are derived from cells not placed in contact with the swimbladder and that they are induced by the primarily induced part of the ectoderm.