Metamorphic change in EP37 expression: members of the ßγ-crystallin superfamily in newt.

EP37 is an epidermis-specific protein found in the developing embryo of the Japanese newt, Cynops pyrrhogaster. Our previous study predicted the presence of genes homologous to EP37, which show temporary shared expression at the turn of metamorphosis. In this study, we isolated and characterized three cDNAs encoding novel EP37 homologues; two from the skin of an adult newt and the other from swimming larva. Conceptual translation of the open reading frames of these cDNAs predicted proteins carrying ßγ-crystallin motifs and putative calcium-binding sites, both of which are features shared by the originally identified EP37 (EP37L1), as well as a spore coat protein of Myxococcus xanthus, protein S. Immunoblot analyses and immunohistochemical studies indicated that two of the EP37 proteins, EP37L1 and EP37L2, are exclusively expressed in the epidermis (skein cells) including the figures of Eberth at premetamorphic stages. During and after metamorphosis, the expression of EP37 proteins was mainly observed in cutaneous glands, and a molecular transition to the adult types of EP37, EP37A1 and EP37A2, occurred. These observations suggest that EP37 proteins play an important role in construction of integumental tissues and adaptation to the aquatic or amphibious environment.

Temporal alteration of dose-dependent response to activin in newt animal-cap explants.

In this study, we examined the dose-dependent responses of animal-pole cells of the Japanese newt, Cynops pyrrhogaster, to activin. Cynops has a slower developmental rate and a simpler animal cap structure than Xenopus. These features enable temporal differences in animal-cap competence to be identified more easily and relatively sharp dose-response profiles can be obtained without cell dissociation. When Cynops caps were excised at the mid-blastula stage and transcript levels of marker genes were examined at the early gastrula stage, the strongest induction of brachyury occurred at a low activin dose, suggesting that cells can recognize changing concentrations of an inducing signal in the embryo. Furthermore, the time course of brachyury expression revealed that caps from the mid-blastula stage exposed to a high dose of activin maintained a low expression level after induction. This suggests that Cynops animal-pole cells can assess activin concentration in a simple and direct manner. In addition, we found that animal-cap competence significantly changes during the blastula stage. The data presented here suggest that this change does not autonomously occur within animal-pole cells but requires signals that emanate from other germ layers.

pax-6 gene expression in newt eye development.

pax-6 is thought to be a master control gene of eye development in species ranging from insects to mammals. We have isolated a pax-6 cDNA homolog of the newt, Cynops pyrrhogaster. RT-PCR and sequence analyses predicted four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shared extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal was first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system - except for the midbrain. The injection of sonic hedgehog (shh) RNA inhibited the expression of pax-6 within the optic vesicle and disturbed eye cup formation. A similar suppressive effect of shh was also observed in the conjugation of the animal caps preloaded with exogenous shh and noggin mRNA, which was used as an inducer of pax-6. In contrast, shh injection had no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Moreover, we found transient activation of pax-6 in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula.

Cloning of an Epidermis-specific Cynops cDNA from Neurula Library.

A cDNA encoding one of the epidermis-specific proteins designated as the spot 6 was isolated from the Cynops embryo. Cynops neurula cDNA library was constructed with the plasmid vector containing the promoter sequence for SP6 RNA polymerase. After transcription and translation in vitro the final protein products were screened for the presence of spot 6 by two-dimensional gel electrophoresis. The total library was digested by 11 restriction endonucleases selected not to destroy the sequence in both the vector and the insert encoding spot 6 protein. The ATP-dependent DNase digestion eliminated the cDNA population sensitive to these endonucleases. These steps effectively enriched the sequence for spot 6 protein. The resultant sublibrary was repeatedly divided into smaller pools and was screened. The tryptic peptide analysis showed that the isolated clone produced the protein identical to the spot 6 protein originally defined in vivo. Northern analyses showed that the cloned gene was expressed as expected from the developmental behavior of the spot 6 in vivo.

Protein Synthesis during Neural and Epidermal Differentiation in Cynops Embryo: (Cynops pyrrhogaster/protein synthesis/neural induction/concanavalin A/epidermal differentiation).

Two-dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with 35 S-methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.

Concanavalin A Acts as a Factor in Establishing the Dorso-Ventral Gradient in the Ventral Mesoderm of Newt Gastrula Embryos1 : (concanavalin A/organizer/dorsalization/dorso-ventral gradient/newt).

Con A induced dorsal differentiation in the ventral mesoderm of Cynops gastrula embryo. This process apparently requires a certain amount of Con A to be internalized as supported by the following evidence: 1) Oligomannose-type oligosaccharide, a potent inhibitor of Con A, considerably inhibited dorsalization of ventral mesoderm by Con A. The incorporation of 125 I-Con A into the ventral mesoderm was greatly inhibited by this sugar. 2) Sepharose-immobilized Con A did not dorsalize the ventral mesoderm. Con A-induced dorsalization was found to be concentration-dependent. Microautoradiograms of 125 I-Con A-treated ventral mesoderm suggest that the target site (some receptor molecules) of Con A exists inside the cell. Con A is the first pure substance reported to mimic the two properties of the organizer-neural induction of the competent ectoderm and dorsalization of the ventral mesoderm. In neural induction, Con A acts on the cell surface, while Con A apparently needs to be internalized to trigger dorsal differentiation. Interestingly, Con A-dorsalized ventral mesoderm acquired the neural inducing function of the organizer within the early phase of dorsalization.