Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells

PlOtp (Orthopedia), a homeodomain-containing transcription factor, has been recently characterized as a key regulator of the morphogenesis of the skeletal system in the embryo of the sea urchin Paracentrotus lividus. Otp acts as a positive regulator in a subset of oral ectodermal cells which transmit short-range signals to the underlying primary mesenchyme cells where skeletal synthesis is initiated. To shed some light on the molecular mechanisms involved in such a process, we begun a functional analysis of the cis-regulatory sequences of the Otp gene. Congruent with the spatial expression profile of the endogenous Otp gene, we found that while a DNA region from −494 to +358 is shown to drive in vivo GFP reporter expression in the oral ectoderm, but also in the foregut, a larger region spanning from −2044 to +358 is needed to give firmly established tissue specificity. Microinjection of PCR-amplified DNA constructs, truncated in the 5′ regulatory region, and determination of GFP mRNA level in injected embryos allowed the identification of a 5′-flanking fragment of 184 bp in length, essential for expression of the transgene in the oral ectoderm of pluteus stage embryos. Finally, we conducted DNAse I-footprinting assays in nuclear extracts for the 184 bp region and detected two protected sequences. Data bank search indicates that these sites contain consensus binding sites for transcription factors.