Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream ofbeta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos

In early Ciona embryos, nuclear accumulation of beta -catenin is most proba bly the first step of endodermal cell specification. If beta -catenin is mi s- and/or overexpressed, presumptive notochord cells and epidermal cells ch ange their fates into endodermal cells, whereas if beta -catenin nuclear lo calization is downregulated. by the overexpression of cadherin, the endoder m differentiation is suppressed, accompanied with the differentiation of ex tra epidermal cells (Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) D evelopment 127, 3009-3020). Subtractive hybridization screens of mRNAs betw een beta -catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential beta -catenin target genes that are re sponsible for endoderm differentiation in Ciona savignyi embryos. We found that a LIM-homeobox gene (Cs-lhx3), an otx homolog (Cs-otx) and an NK-2 cla ss gene (Cs-ttf1) were among beta -catenin downstream genes. In situ hybrid ization signals for early zygotic expression of Cs-lhx3 were evident only i n the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf 1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expresse d again in a set of neuronal cells in the tailbud embryo, while Cs-otx was expressed in the anterior nervous system of the embryo. Expression of all t hree genes was upregulated in beta -catenin overexpressed embryos and downr egulated in cadherin overexpressed embryos. Injection of morpholino oligonu cleotides against Cs-otx did not affect the embryonic endoderm differentiat ion, although the formation of the central nervous system was suppressed. I njection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs result ed in ectopic development of endoderm. differentiation marker alkaline phos phatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injecti on of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-ca dherin mRNA into eggs resulted in the suppression of alkaline phosphatase d evelopment, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued th e alkaline phosphatase development. These results strongly suggest that a L IM-homeobox gene Cs-lhx3 is one of the beta -catenin downstream genes and t hat its early expression in embryonic endodermal cells is responsible for t heir differentiation.