Development 128, 3559-3570 (2001)
© 2001 The Company of Biologists Limited
Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of ß-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos
Yutaka Satou*,
,
Kaoru S. Imai* and
Nori Satoh
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* These authors contributed equally to this work
Author for correspondence (e-mail: yutaka{at}ascidian.zool.kyoto-u.ac.jp)
Accepted June 20, 2001
In early Ciona embryos, nuclear accumulation of ß-catenin is most probably the first step of endodermal cell specification. If ß-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if ß-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied with the differentiation of extra epidermal cells (
Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) Development 127, 3009-3020
). Subtractive hybridization screens of mRNAs between ß-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential ß-catenin target genes that are responsible 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 class gene (Cs-ttf1) were among ß-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 were evident only in 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-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expressed 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 three genes was upregulated in ß-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx did not affect the embryonic endoderm differentiation, although the formation of the central nervous system was suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs resulted in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs resulted in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the ß-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation.
Key words: Ascidians, ß-Catenin, Target genes, Subtraction screening, Cs-lhx3, Cs-otx, Cs-ttf1, Early expression, Endoderm differentiation, Ciona savignyi
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© The Company of Biologists Ltd 2001
