Replacement of maternal 40S rRNA precursor by newly synthesized precursor in early embryos of Xenopus laevis

The amount of intact 40S rRNA precursor was followed by Northern hybridization in the course of the early embryogenesis of wild-type Xenopus laevis and its anucleolate mutant. The total amount of 40S rRNA precursor did not alter appreciably until the midblastula stage, decreased at the late blastula stage, and then increased. In the anucleolate mutant, in which no rRNA synthesis occurs, the 40S rRNA precursor decreased at the late blastula stage and disappeared after the gastrula stage. In the nuclear fraction of the wild type, the 40S rRNA precursor was detectable after the midblastula stage. Therefore, the 40S rRNA precursor in the pre-blastula embryos is maternal and decreases at the late blastula stage. New synthesis of 40S rRNA precursor apparently occurs after the midblastula stage.     

Fate of amplified nucleoli in Xenopus laevis embryos

We have followed the fate of two components of extrachromosomal nucleoli, amplified ribosomal DNA (rDNA) and 7.5 kb precursor rRNA, during early embryogenesis of Xenopus laevis. Other workers have shown that the amount of amplified rDNA accumulated during oogenesis remains unchanged through the 16-cell stage of embryogenesis. Here we show that as embryonic cleavage continues, the amount of amplified rDNA decreases until it is no longer detectable in the early gastrula embryo. In contrast, the amount of 7.5 kb precursor rRNA in eggs, early cleavage stage embryos, or blastula stage embryos is the same as in oocyte nuclei. Since no rRNA synthesis occurs during these early stages, we conclude that the precursor rRNA sequences synthesized in the oocyte are neither processed nor degraded during early development. The amplified rDNA is not replicated in the early embryo even though the chromosomal DNA of the embryo replicates every 30 min during the first 7.5 hr of embryogenesis. When amplified rDNA is purified and then injected into cleaving embryos, however, we find that it is replicated. This finding suggests that some factor(s) prevents the endogenous amplified rDNA from responding to the cellular replication signals. We show that methylation of cytosine in the rDNA is not related to the DNA's capacity for replication in this system since amplified (unmethylated) and chromosomal (methylated) rDNA are both replicated when injected into embryos. The methylation pattern of these rDNAs appears to be maintained after replication in the embryo.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

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.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

Chromatin proteins from normal,vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

六价铬在斜带石斑鱼胚胎中的生物累积及其对igf2、glut2和pparg基因表达的影响

以斜带石斑鱼(Epinephelus coioides)为研究对象,探讨了水中不同浓度梯度六价铬(0、0.20、0.60、1、5、10、20、40、60和80 mg/L)在胚胎发育不同阶段(桑葚期、囊胚期、原肠末期、脑泡形成期和心脏跳动期)的生物累积及其对细胞生长基因igf2和营养代谢基因glut2、pparg的mRNA表达的影响。结果表明,胚胎中Cr⁶⁺生物累积量和吸收率随暴露浓度的增加而增加,且吸收率随胚胎发育时期的延伸而降低。在桑葚期时,斜带石斑鱼胚胎吸收Cr⁶⁺能力最强,是心脏跳动期的20倍。在桑葚期、囊胚期和原肠末期, Cr⁶⁺对胚胎中igf2和glut2基因mRNA表达水平有显著促进作用(P<0.05),但对pparg基因mRNA表达水平有显著抑制作用(P<0.05)。在原肠末期和脑泡形成期, Cr⁶⁺对胚胎中pparg基因mRNA表达水平有显著促进作用(P<0.05)。研究表明水中Cr⁶⁺暴露在斜带石斑鱼胚胎发育过程中有明显的生物累积,且对细胞生...     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33^oC for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tail-bud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37^oC for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximidetreated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development. © 1995 Wiley-Liss, Inc.     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.