Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos

A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8-cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi.     

Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis

Ascidians are primitive chordates. Their fertilized egg develops quickly into a tadpole-type larva, which consists of a small number but distinct types of cells, including those of epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. In addition, the free-swimming and non-feeding larvae metamorphose into sessile and filter-feeding adults. The genome size of Ciona intestinalis is estimated to be about 160 Mb, and the number of genes approximately 15,500. The present Ciona cDNA projects focused on gene expression profiles of fertilized eggs, 32-110-cell stage embryos, tailbud embryos, larvae, and young adults. Expressed sequence tags (ESTs) of the 5'-most end and 3'-most end of more than 3000 clones were determined at each developmental stage, and the clones were categorized into independent clusters using the 3'-end sequences. Nearly 1000 clusters of them were then analyzed in detail of their sequences against a BLASTX search. This analysis demonstrates that, on average, half of the clusters showed proteins with sequence similarities to known proteins and the other half did not show sequence similarities to known proteins. Genes with sequence similarities were further categorized into three major subclasses, depending on their functions. Furthermore, the expression profiles of all of the clusters were analyzed by whole-mount in situ hybridization. This analysis highlights gene expression patterns characteristic to each developmental stage. As a result, the present study provides many new molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. This sequence information will be used for further comparative genome studies to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans. All of the data fully characterized may be viewed at the web site http://ghost.zool.kyoto-u.ac.jp.     

蛋白激酶C_ζ对小鼠受精卵发育早期基因组转录活化的影响

为研究蛋白激酶Cζ (proteinkinaseCζ ,PKCζ)在小鼠受精卵细胞早期发育过程中对胚胎基因组活化影响 ,采用免疫印迹和细胞免疫荧光的方法 ,观察PKCζ的抑制剂对小鼠受精卵 1 细胞期G1和G2 不同时期小鼠受精卵基因组活化的影响 .小鼠 1 细胞期受精卵蛋白激酶C (PKC)的活性不断增加 ,并在G2 期达到最高 .PKC的抑制剂calphostinC可以明显抑制PKC的活性达 4 7% .同时calphostinC对受精卵 1 细胞期基因组的早期活化具有显著的抑制作用 (P <0 0 1) .在小鼠 1 细胞期受精卵的G2 期 ,具有活性的磷酸化PKCζ的含量明显多于G1期和卵母细胞MⅡ期 ,分别比它们高2 7%和 110 % .PKCζ的特异性抑制剂可以抑制受精卵 1 细胞期基因的转录和活化 (P <0 0 5 ) .实验结果表明 ,PKCζ参与了小鼠受精卵基因组早期转录的调控     

蛋白激酶B在小鼠1-细胞期受精卵中活性及表达变化

蛋白激酶B(proteinkinaseB ,PKB)发现于 1991年 ,属于丝 苏氨酸蛋白激酶 .因其激酶活性区的氨基酸序列与蛋白激酶C (proteinkinaseC ,PKC)和蛋白激酶A (proteinkinaseA ,PKA)同源性分别为 73%和 6 8% ,因此命名为PKB ,或PKA和PKC相关激酶(relatedtheAandCkinase ,RACK) [1] .另外 ,PKB被证明为逆转录病毒的癌基因v akt编码的蛋白产物 ,因此PKB又称AKT[2 ] .PKB分子量 6 0kD ,目前已知分为PKBα、β、γ三种 .PKBα广泛存在于机体各组织中 ,其活性受多种信息物质调节 .PKBβ在卵巢癌、胰腺癌细胞中过表达 ,PKBγ在大…     

Synthesis and phosphorylation of uvomorulin during mouse early development.

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 x 10(3) M(r) precursor and 120 x 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.     

Stage-specific gene expression in asynchronous tetraploid mouse embryos formed by fusion of blastomeres and fertilized eggs

Asynchronous tetraploid mouse embryos were generated by electrofusion of fertilized eggs with blastomeres from different cleavage stages. The majority of the cytoplasm was always contributed by the egg. The best development was observed when eggs were fused with 2-cell blastomeres. Both genomes became active in fusion embryos (at least the genes for glucose phosphate isomerase did). Stage-specific protein synthesis seemed to be more adjusted to the developmental stage of the egg's than of the blastomere's genome, but at the 2-cell stage both contributed slightly differently to the protein patterns. Also, the time range of the first appearance of the stage-specific embryonic antigen SSEA-1 was wider in fusion embryos than in controls. It seems that the two genomes are not completely synchronized in these tetraploid embryos, a further indication that, in the mouse, the cytoplasm of fertilized eggs might not be compatible with older embryonic nuclei. Some results were presented at the 83. Jahresversammlung der Deutschen Zoologischen Gesellschaft in Frankfurt, 04.-09.06.1990 Correspondence to: U. Petzoldt     

Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome

Paternal and maternal genomes apparently have complementary roles during embryogenesis in the mouse, and both are essential for development to term. However, there is no direct evidence to show that functional differences between parental genomes remain intact after activation of the embryonic genome at the 2-cell stage. In this study we demonstrate that transfer of paternal or maternal nuclei from early haploid preimplantation embryos back to fertilized eggs from which one pronucleus was removed resulted in development to term, but only if the remaining pronucleus was of the parental type opposite to the donor nucleus. Hence, functional differences between parental chromosomes are heritable and they survive activation of the embryonic genome and probable reprogramming of donor embryonic nuclei by epigenetic factors in the egg cytoplasm.