Distinct expression of two types of Xenopus Patched genes during early embryogenesis and hindlimb development.

Patched (Ptc) is a putative twelve transmembrane domain protein that is both a Hedgehog (Hh) receptor and transcriptional target of Hh. In this study, we isolated Xenopus Ptc cDNAs, Ptc-1 and Ptc-2, and carried out comparative analyses on their expression patterns. The putative Ptc-2 protein has a long C-terminal extension that has similarities in both length and sequence to those of Ptc-1 proteins in mouse, chick and human. In both early embryogenesis and hindlimb development, Ptc-2 expression is restricted to cells that receive a Hh signal, a pattern similar to that of Gli-1. Ptc-1, however, shows a broader distribution, mainly non-overlapping with that of Ptc-2. Despite the difference in their expression patterns, both are induced in animal cap explants synergistically by Shh and Noggin, showing a conserved regulation in their activation mechanisms.

Expression of five novel T-box genes and brachyury during embryogenesis, and in developing and regenerating limbs and tails of newts.

Several T-box genes are considered to play important roles in developing limbs, tails and neural retinae. Five novel T-box genes in the Japanese newt were isolated and their expression was analyzed, together with another T-box gene of brachyury, during embryogenesis and in the developing and regenerating limbs and tail. Four are designated CpTbx2, CpTbx3, CpTbx6R and CpEomesodermin based on molecular phylogenetic analyses, and the other is named CpUbiqT from its ubiquitous expression. While all were expressed during embryogenesis, only four of them (CpTbx2, CpTbx3, CpUbiqT and brachyury) were detected in developing limbs and/or tails. Except for brachyury, they were continuously expressed in normal adult appendages and showed elevated expression levels in regenerating limbs, whereas only CpTbx2 showed significant up-regulation in regenerating tails. Compared with orthologous genes in other species, CpTbx2, CpTbx3 and CpEomesodermin showed several notable differences such as an abundance of maternal transcripts of CpEomesodermin, a unique insertion sequence within the T-box domain of CpTbx2, and a lack of visible expression of CpTbx2and CpTbx3 in the apical ectodermal region of developing limbs. In view of the uniqueness of the newt, these results are discussed with respect to the possibility of their involvement in regeneration.

Lens regeneration in Xenopus is not a mere repeat of lens development, with respect to crystallin gene expression.

The spatio-temporal expression of three crystallin genes (alpha A, beta B1 and gamma) in lenses of Xenopus laevis was studied by in situ hybridization to compare the process of lens formation in embryonic development with that of lens regeneration from cornea that occurs in the tadpole. During embryonic lens development, all three crystallin transcripts were initially detected at the same stage of lens placode formation, and subsequently their signals became restricted to the presumptive lens fiber region. At later stages, the three crystallin genes were expressed in primary and secondary lens fibers, but not in lens epithelium. During lens regeneration, alpha A- and beta B1-crystallin signals were first detected in the presumptive lens fiber region of the lens vesicle. The expression of gamma-crystallin, however, appeared later than the other two crystallin genes and was detected only in morphologically discernible lens fibers. In the later stages of lens regeneration, expression of these crystallins was observed only in the lens fiber region, similar to embryonic lens development. These results reveal that lens regeneration from the inner layer of the outer cornea is not simply a repetition of embryonic lens development, when examined at the level of crystallin gene transcription.

Pax-6 and Prox 1 expression during lens regeneration from Cynops iris and Xenopus cornea: evidence for a genetic program common to embryonic lens development.

Lens regeneration from non-lens ocular tissues has been well documented in amphibians, from the dorsal iris in the newt and from the outer cornea in Xenopus. To understand the early molecular events which govern lens regeneration, we examined the expression of two early marker genes of normal lens development, Pax-6 and Prox 1. In both Cynops (newt) iris and Xenopus cornea, Pax-6 is expressed soon after lentectomy in a region broader than that giving rise to the regenerating lens, indicative of an important role for Pax-6 in determination of the regeneration potential. Then Prox 1 expression begins within the Pax-6-expressing tissue, and these Prox 1-expressing cells give rise to the regenerating lens. This sequence of events also takes place in the lens placode of the embryo, indicating that the presence of the same genetic program operates in both embryonic lens development and lens regeneration, at least partly. In the Cynops iris, Pax-6 expression occurs initially in the entire marginal region of the iris after lentectomy but then becomes restricted to the dorsal region. Further studies are expected to elucidate the mechanism of this long-standing problem of the dorsal-restriction of lens regeneration from the newt iris.

A critical role for the optic vesicle in lens development; a reinvestigation of free lens formation in Cynops pyrrhogaster.

The role of the optic vesicle in lens development was reinvestigated in Cynops pyrrhogaster. To study the necessity for the optic vesicle in early lens development, the optic anlages of stage 17-27 embryos were ablated and the frequency of free lens formation was examined with lens specific markers. Free lens formation was not observed when operations were performed prior to contact between the head surface epidermis and the optic vesicle (stages 17-18). On the contrary, free lens formation occurred in all cases where the optic vesicles were removed after the initiation of lens placode formation in the head surface epidermis (stage 27). However, no lens fiber formation was observed in these free lenses as judged by the absence of lens fiber specific gene expression, namely gamma-crystallin, at stages when secondary lens fiber formation could be found in the control lenses of the unoperated sides. The pattern of expression of alpha A-crystallin in the developing free lens also differed from that of the normally developing lens. This paper is the first report to indicate that the coordinated and sequential expression of crystallin genes are influenced by the optic vesicle; the optic vesicle is required for proper regulation of the alpha A- and gamma-crystallin but not beta B1-crystallin genes.

Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the betagamma-crystallin superfamily.

EP37 family proteins are non-lens members of the betagamma-crystallin superfamily, of which expression is observed in integumental tissues of the Japanese newt, Cynops pyrrhogaster. In the present study, a gene was isolated that has high homology with ep37 and is transcribed mainly in the gastric epithelial cells and hence designated gep. The predicted amino acid sequence of the gep cDNA contains four betagamma-crystallin motifs in the N-terminal half, as is the case in the integumental EP37 proteins. Immunohistochemical analysis showed that GEP protein was mainly localized on the luminal content of the surface mucous cells of the gastric epithelium in both premetamorphic larvae and adults. In addition, GEP protein was also expressed in fundic glands after metamorphosis. Considering the fact that beta- and gamma-crystallins are evolutionarily related to stress-induced proteins, this localization suggests that GEP protein may have an evolutionarily conserved role in protection against physico-chemical stresses, such as physical abrasion and autodigestion, during assimilation.

Hedgehog and patched gene expression in adult ocular tissues.

We analysed the expression of members of the hh gene family in adult ocular tissues of newt, frog and mouse by RT-PCR method. Shh displayed restricted expression in the neural retina that was conserved in each species analyzed. X-bhh, X-chh and mouse Ihh were detected in the iris and in the retinal pigment epithelium, while mouse Dhh was detected additionally in the neural retina and faintly in the cornea. We also found that two types of ptc genes, potential hh targets and receptors, were expressed in these tissues, suggesting the presence of active hh signalling there.

Activation of two Cynops genes, fork head and sonic hedgehog, in animal cap explants.

We have isolated cDNAs of sonic hedgehog (shh) and fork head from Cynops (Japanese newt) embryo. Their expression was investigated in relation to mesoderm induction by activin and basic fibroblast growth factor (bFGF). Different from these homologs in Xenopus, they are activated not only by activin but also by bFGF in animal cap explants, showing a difference of animal pole cells in responsiveness to bFGF between Cynops and Xenopus. We also investigated the involvement of fork head in shh activation. The expression of shh was activated in animal caps which overexpressed either of two Xenopus fork head homologs, pintallavis/XFD-1 or XFKH-1/XFD-1', indicating that fork head up-regulates the transcription of shh in Cynops embryo.

Yields of tubulin paracrystals, vinblastine-crystals, induced in unfertilized and fertilized sea urchin eggs in the presence of D2O.

The effect of D2O on the crystallization of polymerizable tubulin in sea urchin egg cytoplasm was investigated by estimating the yield of "vinblastine (VB)-crystals" by directly measuring the dimensions of the crystals produced and by protein assays of the crystal isolates. The yield of VB-crystals in mature unfertilized eggs was fairly constant; it neither increased nor decreased in the presence of D2O. On fertilization, the yield of crystals decreased markedly as compared with yields from unfertilized eggs; but, the yield was restored to the value for unfertilized eggs when an adequate concentration of D2O was present during incubation. These results are evidence that tubulin molecules in unfertilized sea urchin eggs are in the polymerizable state but become masked and partly unpolymerizable after fertilization and the D2O releases the masked state and converts unpolymerizable tubulin molecules into active and polymerizable state.

Effects of heavy water (D2O) on the length of the mitotic period in developing sea urchin eggs.

The sequence of mitosis in sea urchin eggs was investigated in the presence and absence of D2O. Direct observations of living cells under a polarizing microscope and observations with fixation-staining procedures were used. The duration of mitosis was extended by the presence of D2O. The slight extension of anaphase was due to elongation of the spindle in D2O, but the period from prophase to metaphase was clearly prolonged in the deuterated condition. These results indicate that D2O does not suppress anaphase chromosome movement, but does affect prometaphase and delays the alignment of chromosomes on the equatorial plane of the mitotic spindle at metaphase. The stability of the isolated mitotic apparatus against Ca ions and low temperature also was investigated. There was no difference in the deterioration of isolated spindle birefringence under normal and deuterated conditions. The implications of these results are discussed in relation to the enhancement effect of D2O on the volume and birefringence of the living mitotic spindle.