The Otx binding site is required for the activation of HpOtxL mRNA expression in the sea urchin, Hemicentrotus pulcherrimus.

Two distinct types of orthodenticle-related (HpOtxE and HpOtxL) mRNA are transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing, and their expressions are differentially regulated during early development of the sea urchin Hemicentrotus pulcherrimus. To understand the mechanism of this regulation, we screened for the enhancer element involved in the stage-specific expression of HpOtxL mRNA. Different portions of the HpOtx gene, including the 5'-flanking region and the first intron, were ligated to the minimal HpOtxL promoter driving a luciferase gene, and their constructs were introduced into fertilized eggs using a particle gun. The enhancer element responsible for proper expression consistent with that of the endogenous HpOtxL was found in the first intron of the HpOtx gene. External and internal deletion analyses showed that the 334 bp region (from +8838 bp to +9171 bp) was required for enhancer activity. In addition, deletion of an Otx binding site within the 334 bp region markedly reduced reporter expression. These results suggest that the Otx binding site within the HpOtxL enhancer is required for the activation of HpOtxL mRNA expression. The promoter preference of the HpOtxL enhancer is also discussed.