The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymalprogeny of four precursor cells, the micromeres, which are determined to theskeletogenic pathway by a process known as cytoplasmic localization. A geneencoding one of the major products of the skeletogenic mesenchyme, a prominent50 kD protein of the spicule matrix, has been characterized in detail. cDNA cloneswere first isolated by antibody screening of a phage expression library, followed byisolation of homologous genomic clones. The gene, known as SM50, is single copy inthe sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressedonly in skeletogenic cells. Transcripts are first detectable at the 120 cell stage,shortly after the segregation of the skeletogenic precursors from the rest of theembryo. The SM50 open reading frame begins within the first exon, is 450 amino acidsin length, and contains a loosely repeated 13 amino acid motif rich in acidic residueswhich accounts for 45% of the protein and which is possibly involved in interactionwith the mineral phase of the spicule.
The important cis-acting regions of the SM50 gene necessary for properregulation of expression were identified by gene transfer experiments. A 562 bppromoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50first exon (including the SM50 initiation codon), was both necessary and sufficient todirect high levels of expression of the bacterial chloramphenicol acetyltransferase(CAT) reporter gene specifically in the skeletogenic cells. Removal of promotersequences between positions -2200 and -438, and of transcribed regions downstream of+124 (including the SM50 intron), had no effect on the spatial or transcriptionalactivity of the transgenes.
Regulatory proteins that interact with the SM50 promoter were identified by thegel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins.Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one isrequired for expression. Two additional sites are also present in the promoter of theaboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA Telement, the other a putative repressor element. The fifth site overlaps the bindingsite of the putative repressor and may function as a positive regulator by interferingwith binding of the repressor. All of the proteins are detectable in nuclear extractsprepared from 64 cell stage embryos, a stage just before expression of SM50 isinitiated, as well as from blastula and gastrula stage; the putative enhancer bindingprotein may be maternal as well.
