Two Isoforms of Orthodenticle-Related Proteins (HpOtx) Bind to the Enhancer Element of Sea Urchin Arylsulfatase Gene

Abstract

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation of HpArs gene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms.

A Sea Cucumber Homolog of the Mouse T-Brain-1 is Expressed in the Invaginated Cells of the Early Gastrula in Holothuria leucospilota.

“…HpArs (Sakamoto et al, 1997;Kiyama et al, 1998), Spec2a (Gan et al, 1995) and Endo16 (Yuh et al, 1998) have been reported as direct target genes of Otx in sea urchin embryo. HpArs is transcriptionally activated late in blastula, and after the gastrula stage it is expressed exclusively in the aboral ectoderm throughout embryonic development (Sasaki et al, 1988;Akasaka et al, 1990aAkasaka et al, , 1990b.…”

“…In zebrafish three types of Otx are present in the developing diencephalon and midbrain (Mori et al, 1994). In sea urchin development, two isoforms of Otx are expressed (Mao et al, 1994;Sakamoto et al, 1997). The first type of Otx protein, referred to as an early-type Otx (HpOtxE), appears in early development and gradually decreases in amount by the gastrula stage.…”

“…The second type of Otx protein, referred to as a late-type of Otx (HpOtxL), appears at the blastula stage and remains until gastrula stage. The nucleotide sequence reveals that the homeobox and downstream regions through to the C-terminus are identical in the two types of HpOtx proteins, while the N-terminal region has different polypeptides (Sakamoto et al, 1997) (Fig. 4a).…”

“…This region contains a tandem repeat of core consensus sequences of orthodenticlerelated protein (Otx) binding sites, which serve as the major source of positive control for HpArs gene (Sakamoto et al, 1997) (Fig. 6a).…”

“…We assumed that a boundary of chromatin is located in the flanking region of such an actively transcribed Ars gene. In order to find putative insulator elements in the upstream region of Ars, we used the well-characterized Ars enhancer C15 (225 bps), which contains Otx binding sites as major positive elements Sakamoto et al, 1997;Kiyama et al, 1998), together with Ars promoter (-252 bp to +38 bp) Morokuma et al, 1997) to drive a luciferase reporter gene. The constructs were introduced in the fertilized eggs by particle gun and the transcriptional activity was determined at gastrula stage when the Ars gene is actively transcribed.…”