Action of morpholinos in Ciona embryos

First, using morpholino against lacZ, we demonstrate that the morpholino specifically suppresses the translation of the gene introduced exogenously into Ciona eggs. Second, using morpholino against an alkaline phosphatase gene, we show that the morpholino suppresses the translation of the endogenous gene as well. Third, using morpholino against beta-catenin gene, we confirm that the suppression by the morpholino can be rescued by injection of beta-catenin mRNA. All of these results indicate that morpholino act in Ciona embryos to specifically block the function of endogenous genes as well as exogenously introduced genes. genesis 30: 103--106, 2001.     

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.     

Isolation and characterization of beta-catenin downstream genes in early embryos of the ascidian Ciona savignyi

Nuclear localization of beta-catenin is most likely the first step of embryonic axis formation or embryonic cell specification in a wide variety of animal groups. Therefore, the elucidation of beta-catenin target genes is a key research subject in understanding the molecular mechanisms of the early embryogenesis of animals. In Ciona savignyi embryos, nuclear accumulation of beta-catenin is the first step of endodermal cell specification. Previous subtractive hybridization screens of mRNAs between beta-catenin-overexpressed embryos and nuclear beta-catenin-depleted embryos have resulted in the identification of beta-catenin downstream genes in Ciona embryos. In the present study, I characterize seven additional beta-catenin downstream genes, Cs-cadherinII, Cs-protocadherin, Cs-Eph, Cs-betaCD1, Cs-netrin, Cs-frizzled3/6, and Cs-lefty/antivin. All of these genes were expressed in vegetal blastomeres between the 16-cell and 110-cell stages, although their spatial and temporal expression patterns were different from one another. In situ hybridizations and real-time PCR revealed that the expression of all of these genes was up-regulated in beta-catenin-overexpressed embryos, and down-regulated in beta-catenin-suppressed embryos. Therefore, the accumulation of beta-catenin in the nuclei of vegetal blastomeres activates various vegetally expressed genes with potentially important functions in the specification of these cells.     

Beta-catenin is required for the establishment of vegetal embryonic fates in the nemertean, Cerebratulus lacteus

Downstream components of the canonical Wnt signaling pathway that result in the nuclear localization of beta-catenin are involved in diverse developmental processes including the formation of the mesendoderm, the regulation of axial properties and asymmetric cell divisions in a wide array of metazoans. The nemertean worm, Cerebratulus lacteus, represents a member of the understudied lophotrochozoan clade that exhibits a highly stereotyped spiral cleavage program in which ectodermal, endodermal, and mesodermal origins are known from intracellular fate mapping studies. Here, the embryonic distribution of beta-catenin protein was studied using injection of synthetic mRNA, encoding GFP-tagged beta-catenin, into fertilized eggs. During the early cleavage stages beta-catenin was destabilized/degraded in animal hemisphere blastomeres and became localized to the nuclei of the four vegetal-most cells at the 64-cell stage, which give rise to definitive larval and adult endoderm. Functional assays indicate that beta-catenin plays a key role in the development of the endoderm. Morpholino knockdown of endogenous beta-catenin, as confirmed by Western analysis, resulted in the failure to gastrulate, absence of the gut and an animalized phenotype in the resulting larvae, including the formation of ectopic (anterior) apical organ tissue with elongated apical tuft cilia and no indications of dorsoventral polarity. Similarly, over-expression of the cytoplasmic domain of cadherin or a beta-catenin-engrailed repressor fusion construct prevented endoderm formation and generated the same animalized phenotype. Injections of mRNA encoding either a stabilized, constitutively activated form of beta-catenin or a dominant negative form of GSK3-beta converted all or nearly all cells into endodermal fates expressing gut-specific esterase. Thus, beta-catenin appears to be both necessary and sufficient to promote endoderm formation in C. lacteus, consistent with its role in endoderm and endomesoderm formation in anthozoan cnidarians, ascidians, and echinoderms. Consistent with the results of other studies, beta-catenin may be viewed as playing a role in the development of posterior/vegetal larval fates (i.e., endoderm) in C. lacteus. However, unlike the case found in polychaete annelid and soil nematode embryos, there is no evidence for a role of beta-catenin in regulating cell fates and asymmetric cell divisions along the entire anterior-posterior axis.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Synergistic action of HNF-3 and Brachyury in the notochord differentiation of ascidian embryos.

In vertebrate embryos, the class I subtype forkhead domain gene HNF-3 is essential for the formation of the endoderm, notochord and overlying ventral neural tube. In ascidian embryos, Brachyury is involved in the formation of the notochord. Although the results of previous studies imply a role of HNF-3 in notochord differentiation in ascidian embryos, no experiments have been carried out to address this issue directly. Therefore the present study examined the developmental role of HNF-3 in ascidian notochord differentiation. When embryos were injected with a low dose of HNF-3 mRNA, their tails were shortened and when embryos were injected with a high dose of HNF-3 mRNA, which was enough to inhibit differentiation of epidermis and muscle, no obvious ectopic differentiation of endoderm or notochord cells was observed. However, co-injection of HNF-3 mRNA along with Brachyury mRNA resulted in ectopic differentiation of notochord cells in the animal hemisphere, suggesting that HNF-3 acts synergistically with Brachyury in ascidian notochord differentiation. Notochord differentiation of the A-line precursor cells depends on inducing signal(s) from endodermal cells, which can be mimicked by bFGF treatment. Treatment of notochord precursor cells isolated from the 32-cell stage embryoswith bFGF resulted in upregulation of both the HNF-3 and Brachyury genes.     

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.     

Ultraviolet-sensitive ooplasmic factors are responsible for the development of an endoderm-specific alkaline phosphatase in the ascidian embryo

The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue-specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m²) of ultraviolet (UV) light did not eliminate the AP-promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle-marker. A higher dose (4500 J/m²) of UV light markedly reduced the AP-promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants.