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Eight restriction fragments (I-VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus, and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between -2.8 kb and -2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis-acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.

To define the cis-acting regulatory elements required for proper ontogenic expression of the Arylsulfatase gene in the sea urchin (Hemicentrotus pulcherrimus) embryo, we have constructed the fusion genes composed of bacterial CAT gene and various regions from the 5' flanking sequence of the Ars gene, injected them into unfertilized sea urchin eggs and monitored their expression during development. A wild type construct containing the 5' flanking region of the Ars gene spanning from -2 to -3160 was expressed under proper temporal regulation following injection into unfertilized eggs. By deletion analysis the transcriptional enhancers were localized between -1280 and -2680 of the Ars gene. In addition, sequences acting as a negative regulator as well as a minimal promoter for expression of the Ars gene were detected by internal deletion analysis.

A marked increase in the Na + , K + -ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na + , K + -ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na + , K + -ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na + , K + -ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8-16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li + than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na + , K + -ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.

Previously we showed that the arylsulfatase (Ars) activity increases markedly after the mesenchyme blastula stage, and that the increase in its activity is due to initiation of the Ars gene transcription. In order to detect cells responsible for Ars mRNA production in the sea urchin embryo, we carried out in situ hybridization with the use of 35 S-labeled Ars RNA probe transcribed from Ars cDNA in a vector pT7T3-19U. The Ars gene is uniformly expressed in the cells of blastula, and the expression becomes restricted to aboral ectoderm during development by gastrula stage.