Two Otx proteins generated from multiple transcripts of a single gene in Strongylocentrotus purpuratus

Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin, Strongylocentrotus purpuratus, an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from 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 SpOtx gene. SpOtx(alpha) and SpOtx(beta) had identical C-termini and homeoboxes 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 contrast, three distinct SpOtx(beta) mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(beta) appeared later in development than 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, whereas SpOtx(beta) had no effect on the accumulation of SpOtx(alpha) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.