Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.     

Gastrulation of Gastrotheca riobambae in comparison with other frogs

Blastopore formation, the embryonic disk, archenteron and notochord elongation, and Brachyury expression in the marsupial frog Gastrotheca riobambae was compared with embryos of Xenopus laevis and of the dendrobatids Colostethus machalilla and Epipedobates anthonyi. In contrast with X. laevis embryos, the blastopore closes before elongation of the archenteron and notochord in the embryos of G. riobambae and of the dendrobatid frogs. Moreover, the circumblastoporal collar (CBC) thickens due to the accumulation of involuted cells. An embryonic disk, however, is formed only in the G. riobambae gastrula. We differentiate three gastrulation patterns according to the speed of development: In X. laevis, elongation of the archenteron and notochord begin in the early to mid gastrula, whereas in the dendrobatids C. machalilla and E. anthonyi the archenteron elongates at mid gastrula and the notochord elongates after gastrulation. In G. riobambae, only involution takes place during gastrulation. Archenteron and notochord elongation occur in the post gastrula. In the non-aquatic reproducing frogs, the margin of the archenteron expands anisotropically, resulting in an apparent displacement of the CBC from a medial to a posterior location, resembling the displacement of Hensen's node in the chick and mouse. The differences detected indicate that amphibian gastrulation is modular.     

Participation of proteasomes in Xenopus embryogenesis

We examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.     

Development of the dendrobatid frog Colostethus machalilla

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.     

Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis.

We have designed antisense oligodeoxyribonucleotides which are both highly resistant to nucleolytic degradation and also serve as substrates for ribonuclease H. Using these compounds we have targeted the specific degradation of several maternal mRNAs present in Xenopus laevis oocytes and early embryos. Several internucleoside linkages at both the 3' and 5' ends of the oligonucleotides were modified as phosphoramidates to provide complete protection against exonucleases, the predominant nucleolytic activity found in both oocytes and embryos. Eight Internal linkages were left unmodified to provide a substrate for RNase H. Degradation of specific embryonic mRNAs was accomplished using subtoxic amounts of the modified oligonucleotides. Specific depletion of An2, a localized mRNA encoding the alpha subunit of the mitochondrial ATPase, produced embryos that gastrulated later than control embryos and arrested in development prior to neurulation. A modified oligonucleotide targeting Xenopus cyclin B1 and cyclin B2 mRNA was also synthesized. Following the injection of one blastomere of a two-cell embryo with the anti-cyclin oligonucleotide, cell division in that half of the embryo was inhibited, demonstrating the in vivo importance of these cyclins in mitosis. The oligonucleotide analogs described here should be useful in studying developmentally significant proteins in Xenopus.     

Inhibition of proteoglycan synthesis eliminates left-right asymmetry in Xenopus laevis cardiac looping

The heart of any vertebrate is formed from an apparently symmetric cardiac tube that loops consistently in the same direction along the left-right axis of the embryo. In the amphibian Xenopus laevis, inhibition of proteoglycan synthesis by p-nitrophenyl-beta-D-xylopyranoside during a narrow period of development from late gastrula to early neurula specifically eliminated the looping of the cardiac tube. Most of the proteoglycans synthesized during this period were heparan sulfate proteoglycans. Treatment with p-nitrophenyl-alpha-D-xylopyranoside, an analogue that does not inhibit proteoglycan synthesis, did not interfere with cardiac looping. The critical period for proteoglycan synthesis was coincident with the migration of cardiac primordia to the ventral midline. The inhibition of cardiac looping was further explored in explants of cardiac primordia and anterioventral ectoderm. In recombinate embryos in which half the embryo, and thus one of the two heart primordia, was treated with p-nitrophenyl-beta-D-xylopyranoside, and the other half was untreated, cardiac looping occurred normally. It is proposed that the left-right axis in Xenopus, as reflected in cardiac looping, is established early in development, and that proteoglycan synthesis is involved in the transduction of left-right axial information to the cardiac primordia during migration.     

Wound healing ability of Xenopus laevis embryos. I. Rapid wound closure achieved by bisectional half embryos

We examined wound closure in 'half embryos' produced by the transverse bisection of Xenopus laevis embryos at the primary eye vesicle stage. Both the anterior- and posterior-half embryos survived for more than 6 days, and grew into 'half tadpoles'. Histology and videomicroscopy revealed that the open wound in the half embryo was rapidly closed by an epithelial sheet movement in the wound marginal zone. The time-course of wound closure showed a downward convex curve: the wound area decreased to one-fifth of the original area within 30 min, and the wound continued to contract slowly thereafter. The rapidity of closure of the epidermis as well as the absence of inflammatory cells are typical features of an embryonic type of wound healing. There was a dorso-ventral polarity in the motility of the epidermis: the wound was predominantly closed by the ventral and lateral epidermis. The change in the contour of the wound edge with time suggested a complex mechanism involved in the wound closure that could not be explained only by the purse-string theory. The present experimental system would be a unique and useful model for analyses of cellular movements in the embryonic epithelia.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Antioxidant metabolism of Xenopus laevis embryos during the first days of development

Reactive oxygen species (ROS) are formed and degraded in all aerobic organisms, but their role during embryonic development has not yet been well established. In this paper, we report the activities of various enzymes involved in antioxidant metabolism during the first 7 days of embryonic development of Xenopus laevis embryos. During the first two days of development, embryo antioxidant metabolism is based on catalase and superoxide dismutase activities. Later, the glutathione system is activated, and the activity of all the enzymes involved increases. The results presented in this study, together with previously reported data, support the hypothesis that antioxidant defences may include enzymes that are genetically regulated, while the other systems that appear to be environmentally modulated become relevant later in development, probably to protect embryos from environmental and toxic factors.     

Role of c-myc protein in the early embryonic development of Xenopus

Microinjections of antibodies directed against the protein encoded by the c-myc protooncogene strongly inhibit or arrest the early cell cleavage stage of Xenopus laevis embryos. Injections in one blastomere of a two cell stage embryo inhibit the segmentation of this blastomere. The cleavage of the uninjected blastomere behaves normally. Injections of control rabbit immunoglobulins do not alter the embryonic development.