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 184bp 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 184bp region and detected two protected sequences. Data bank search indicates that these sites contain consensus binding sites for transcription factors.
Cavalieri
Vincenzo
V
Dipartimento di Biologia Cellulare e dello Sviluppo A. Monroy, Università di Palermo, Viale delle Scienze Parco d'Orleans II, 90128 Palermo, Italy.
Bernardo
Maria Di
MD
Spinelli
Giovanni
G
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004064:Digestive System / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D004475:Ectoderm / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D047328:Paracentrotus / Q000166:cytology / Q000196:embryology* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics
D018398:Homeodomain Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA