2 OTX PROTEINS GENERATED FROM MULTIPLE TRANSCRIPTS OF A SINGLE-GENE IN STRONGYLOCENTROTUS-PURPURATUS

Orthodenticle-related (Otx) proteins are a highly conserved class of h omeobox-containing transcription factors found in a wide range of orga nisms. They function in numerous developmental events, most prominentl y, anterior head patterning in insects and vertebrates. In the sea urc hin, Strongylocentrotus purpuratus, an orthodenticle-related protein c alled SpOtx is believed to direct the activation of the aboral ectoder m-specific Spec2a gene and more generally the differentiation of abora l ectoderm cells. To learn more about the structure, expression, and f unction of SpOtx and compare its properties with those of orthologs fr om other species, we isolated cDNA and genomic clones containing SpOtx sequences. Here, we report that SpOtx exists in two forms (alpha and beta) that are generated by alternative RNA splicing from a single SpO tx gene. SpOtx(alpha) and SpOtx(beta) had identical C-termini and home oboxes but were entirely different in their N-terminal domains. SpOtx( alpha) mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contra st, three distinct SpOtx(beta) mRNAs resulted from two separate transc riptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later wer e largely restricted to oral ectoderm and vegetal, plate territories. DNA-binding activity for SpOtx(beta) appeared later in development tha n SpOtx(alpha). Overexpression of SpOtx(alpha) and SpOtx(beta) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(alpha ) was able to repress the accumulation of SpOtx(beta) transcripts, whe reas SpOtx(beta) had no effect on the accumulation of SpOtx(alpha) tra nscripts. These results demonstrate that novel forms of Otx are produc ed in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to divers e forms of Otx in vertebrates. (C) 1997 Academic Press.