A FOX Binding Site is Necessary for the Activation of Pl-Tuba1a Gene in the neurogenic domains

All echinoderm larvae possess a nervous system consisting of a ciliary band and associated sensory ganglia (apical, oral and lateral ganglia) that controls swimming and feeding. Neurons of the larval nervous system first appear as neuroblasts in the thickened ectoderm of the animal plate (anterior neuroectoderm, ANE) at the late blastula – early gastrula stage and then also in the ciliary band. The neural differentiation process of sea urchin embryos has been analysed and the Gene Regulatory Network involved in the differentiation processes is extensively studied. We have previously isolated an alpha tubulin family member of the sea urchin Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the neurogenic territory of embryo (1). In order to identify cis-regulatory elements controlling its spatiotemporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the neurogenic territory of the embryo. The functional analysis of the ICR3 showed that 10 bp (corresponding to a putative forkhead box consensus sequence binding site) are necessary for the gene activation. Additionally, the point mutation of this site in ICR3 prevents Pl-Tuba1a expression.