Ascidian embryos sequester a specific cytoplasm, called the postplasm, at the posterior pole, where many maternal RNAs and proteins accumulate. Although the postplasm is thought to act as the germ plasm, it is also highly enriched in several factors essential for somatic cell development, and how the postplasm components regulate both germ and somatic cell differentiation remains elusive. Using a vasa homolog, CiVH, and other postplasmic components as markers, we found that the postplasmcontaining blastomeres, the B7.6 cells, undergo an asymmetric cell division during gastrulation to produce two distinct daughter cells: B8.11 and B8.12. Most of the postplasmic components segregate only into the B8.11 cells, which never coalesce into the gonad. By contrast, the maternal CiVH RNA and protein are specifically distributed into the B8.12 cells, which divide further and are incorporated into the gonad in juveniles. In the B8.12 cells, CiVH production is upregulated from the maternal RNA source, resulting in the formation of perinuclear CiVH granules, which may be the nuage, a hallmark of germ cells in many animal species. We propose that the redistribution of specific maternal molecules into the B8.12 cells is essential for germ-cell specification in ascidians.

Many kinds of animal embryos exhibit stereotyped cleavage patterns during early embryogenesis. In the ascidian Halocynthia roretzi, cleavage patterns are invariant but they are complicated by successive unequal cleavages that occur in the posterior region. Here we report the essential roles of a novel structure, called the centrosome-attracting body (CAB), which exists in the posterior pole cortex of cleaving embryos, in generating unequal cleavages. By removing and transplanting posterior egg cytoplasm and by treatment with sodium dodecyl sulfate, we demonstrated that loss of the CAB resulted in abolishment of unequal cleavage, while ectopic formation of the CAB caused ectopic unequal cleavages to occur. Experiments with a microtubule inhibitor demonstrated that the centrosome and nucleus were attracted toward the posterior cortex, where the CAB is located, by shortening of microtubule bundles formed between the centrosome and the CAB. Consequently, the mitotic apparatus was positioned asymmetrically, resulting in unequal cleavage. Immunohistochemistry provided evidence that a microtubule motor protein, a kinesin or kinesin-like molecule, may be associated with the CAB. Formation of the CAB during the early cleavage stage was resistant to treatment with the microtubule inhibitor. In contrast, the integrity of the CAB was lost upon treatment with a microfilament inhibitor. We propose that the CAB plays key roles in the orientation and positioning of cleavage planes during unequal cell division.

No abstract

The mechanism of unequal cleavage is one of the most intriguing subjects in cell biology. Previous studies of unequal cleavage have focused on a limited number of organisms such as yeasts, nematodes, sea urchins and annelids. The cleavage pattern of the ascidian embryo is invariant. In the ascidian embryo, the posterior-most blastomeres divide unequally in three successive cleavages. In the present study, it was shown that the ascidian embryo provides another good experimental system with which to analyze the mechanism of unequal cleavage. A novel structure, designated as CAB (centrosome-attracting body), which was found specifically in the unequally cleaving blastomeres was described. In the course of unequal cleavages, first, a thick microtubule bundle appeared between CAB and one of the centrosomes. Then with the shortening of the microtubule bundle, the nucleus with the centrosome was drawn toward CAB, situated at the posterior cortex of the blastomere. Finally, a cleavage furrow formed in the middle of the asymmetrically located mitotic apparatus and produced two blastomeres of different size, generating a smaller cell that inherits CAB. The CAB seemed to play an essential role in the unequal cleavages in the ascidian embryo.

A set of 1,378 expressed sequence tags (ESTs), both the 5'-most and 3'-most ends, derived from Ciona intestinalis fertilized eggs was categorized into 1,003 independent clusters. When compared with sequences in databases, 452 of the clusters showed significant matches with reported proteins, while 190 showed matches with putative proteins for which there is not enough information to categorize their function, and 361 had no significant similarities to known proteins. Sequence similarity analyses of the 452 clusters in relation to the biological function as well as the structure of the message population at this stage demonstrated that 362 of them have functions that many kinds of cells use, 65 are associated with cell-cell communication, including a candidate cDNA for sonic hedgehog, and 25 are transcription factors. Sequence prevalence distribution analysis demonstrated that the great majority (78%) of the mRNAs are rare mRNAs or are represented by a single clone/cluster. All of the 1,003 clusters were subjected to whole-mount in situ hybridization to analyze the distribution of the maternal mRNAs in fertilized eggs, and a total of 329 genes showed localized distribution of the mRNAs: 16 showed cortical localization, 12 showed mitochondrial-like distribution, 99 crescent-like distribution, 63 partial localization, and 139 weak localization. When the distribution pattern of all the maternally expressed mRNAs was examined in the 8-cell stage embryos, it became evident that 248 genes which have localized mRNA patterns at the fertilized egg stage lose their localized distribution by the 8-cell stage. In contrast, 13 genes newly gain a localized pattern by the 8-cell stage. In addition, a total of 39 genes showed distinct in situ signals in the nucleus of blastomeres of the 8-cell stage embryo, suggesting early zygotic expression of these genes by this stage. These results suggest that complicated cytoplasmic movements are associated with the characteristic distribution of maternal mRNAs, which in turn support proper embryonic axis formation and establishment of the genetic network for embryonic cell specification.