Correct expression of spec2a in the sea urchin embryo requires both Otx and other cis-regulatory elements

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16 in the vegetal plate and f or the activation of spec2a in the aboral ectoderm. Because Otx binding sit es alone do not appear to be responsible for the spatially restricted expre ssion of spec2a, additional DNA elements were sought. We show here that con sensus Otx binding sites fused to basal promoters are sufficient to activat e CAT reporter gene expression in all tell types, although expression in en domesoderm progenitors is enhanced. On the other hand, three non-Otx elemen ts derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates gener al activation in the ectoderm. A second element lying between the Otx and Y /CBF sites, called OER, functions to prevent expression in the oral ectoder m. A third site, called ENR, overlapping another Otx site, is required to r epress endoderm expression. Three distinct DNA binding proteins interact se quence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhance r thus consists of closely linked activator and repressor elements that fun ction collectively to cause expression of the spec2a gene in the aboral ect oderm. (C) 2001 Academic Press.