Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.     

Dissection of the XChk1 signaling pathway in Xenopus laevis embryos

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints, xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 in Xenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.     

Gene Expression from Endogenou and Exogenously-introduced DNAs in Early Embryogenesis of Xenopus laevis

In Xenopus laevis embryogenesis, gene expression from endogenous and exogenously-introduced DNAs is reported to start only after 12 rounds of cleavage, at the stage called midblastula transition (MBT). In isotopic labeling experiments, however, we found that the synthesis of heterogeneous mRNA-like RNA occurs from the early cleavage stage, and that gene expression from zygotic genomes occurs sequentially in three characteristically different phases: the pre-MBT phase, in which heterogeneous mRNA-like RNA and small amounts of small-molecular-weight RNAs are synthesized; the MBT phase, in which there is a large increase in 4S RNA synthesis, and the post-MBT phase, in which rRNA is also synthesized. Moreover in our studies on the expression of exogenously introduced DNA, we found that circular forms of bacterial chloramphenicol acetyltransferase (CAT) genes connected to viral promoters were expressed from the early cleavage stage, whereas circular forms of genes connected to Xenopus cardiac α-actin promoter were expressed only after the embryos reached the neurula stage, when the endogenous α-actin gene started to be expressed. We, therefore, conclude that in Xenopus embryogenesis, DNA-dependent RNA polymerases II, III and I are activated in this order, and that the promoter, not changes associated with the MBT, probably determine the timing of gene expression.     

The midblastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.     

In contrast to other Xenopus genes the estrogen-inducible vitellogenin genes are expressed when totally methylated

The methylation-sensitive restriction enzymes Hha I and Hpa II were used to analyze the methylation pattern of four Xenopus laevis genes in DNA of embryos, of erythrocytes, and of untreated and estrogen-treated hepatocytes. Within these four genes all sites tested are fully modified in embryonic DNA. However, the adult β1-globin gene is unmethylated in DNA of erythrocytes, where it is expressed, and the 68 kd albumin gene, active only in hepatocytes, is specifically hypomethylated in hepatic DNA. The vitellogenin genes A1 and A2, in hepatocytes simultaneously expressed upon estrogen treatment, are heavily methylated in all adult tissues, irrespective of expression. Our results reveal that specific genes can be actively transcribed even when they are fully methylated and that changes in the methylation pattern are not a general prerequisite for gene activation.     

Tissue-specific developmental expression of OAX, a Xenopus repetitive element

Approximately 1% of the Xenopus laevis genome consists of highly repetitive DNA known alternatively as OAX (for Oocyte Activation in Xenopus), Satellite I, or Repetitive HindIII Monomer 2. Present as tandemly repeated units of approximately 750 base pairs, OAX encodes a family of small RNA species transcribed by RNA polymerase III. Although the subject of many of the classic studies on early embryonic gene regulation, reports on OAX expression remain contradictory and incomplete. Using whole-mount in situ hybridization and RNase protection assays, we have therefore examined in detail the expression pattern of OAX in Xenopus embryos of various stages. OAX is initially expressed during gastrula stages; by tailbud stages embryos display discrete zones of expression at the dorsal boundary of the cement gland, in the developing somites and differentiating skeletal muscle, as well as in the dorsal aspect of the neural tube. These data demonstrate that OAX is expressed in a dynamic pattern under tight spatial and temporal regulation.     

Marked Alteration at Midblastula Transition in the Effect of Lithium on Formation of the Larval Body Pattern of Xenopus laevis

Embryos of Xenopus laevis were exposed to 0.4 M LiCl for 5 min at various stages of development. The effect of lithium on the larval body pattern could be detected from the 2-cell to the late gastrula stage, but changed from reduction of posterior structures ("anteriorization") to reduction of anterior structures ("posteriorization") just after the 12th cleavage, the time of midblastula transition (MBT). Temporal coincidence of MBT with alteration of the effect of lithium was observed even with embryos derived from half-egg fragment, in which MBT occurs just after the 11th cleavage. These results suggest the existence of a mechanism for formation of the basic plan of the larval body whose function changes at MBT. Combinations of the pre- and post-MBT exposures to lithium induced marked posteriorization in most larvae, indicating that the basic plan is not irreversibly determined until MBT, but is fixed during post-MBT stages.     

Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer

Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.