Two Otx Proteins Generated from Multiple Transcripts of a Single Gene inStrongylocentrotus purpuratus

Abstract

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.

Expression of an Otx gene in the adult rudiment and the developing central nervous system in the vestibula larva of the sea urchin Holopneustes purpurescens.

“…The two forms of protein are identical in the homeodomains and C terminal regions but differ in the N terminal regions. The SpOtx(β) form has similarities with the vertebrate Otx1 and Otx2 gene products (Li et al, 1997). The cDNA sequence of the Otx gene we report for H. purpurescens, HprOtx, aligns with the cDNA of SpOtx(α) and HpOtx E .…”

“…1 with the amino acid sequences of the complete coding regions of SpOtx(α) (Gan et al, 1995;Li et al, 1997) and HpOtx E (Sakamoto et al, 1997). The alignment is divided into two regions: an N terminal region which characterizes the α protein isoform of SpOtx and the E protein isoform of HpOtx, then a C terminal region which is common to both the α and β isoforms of SpOtx (Li et al, 1997) and the E and L isoforms of HpOtx (Sakamoto et al, 1997). This common C terminal region includes the homeodomain.…”

“…In sea urchins, Otx genes have been characterized in Strongylocentrotus purpuratus (Li et al, 1997) and Hemicentrotus pulcherrimus (Kiyama et al, 1998). Two proteins, SpOtx(α) and SpOtx(β), are generated from a single gene in S. purpuratus (Li et al, 1997) and correspond respectively to the early and late proteins, HpOtx E and HpOtx L in H. pulcherrimus, also generated from a single gene (Kiyama et al, 1998). The two forms of protein are identical in the homeodomains and C terminal regions but differ in the N terminal regions.…”

Repression of mesodermal fate byfoxa, a key endoderm regulator of the sea urchin embryo

“…Furthermore, in the sea urchin embryo, 80% of all regulatory genes are used just to get to the late gastrula stage (Howard-Ashby et al, 2006), and multiple re-use of these genes is therefore an inescapable inference. Many particular examples of regulatory genes that execute totally distinct functions during sea urchin embryogenesis are indeed already in hand, for example hnf6 (Otim et al, 2004), otx (Li et al, 1997;Yuh et al, 2002), deadringer (Amore et al, 2003), gsc (Angerer et al, 2001), and blimp1/krox (Livi and Davidson, 2006a;Livi and Davidson, 2006b). The C. elegans ortholog of the foxa gene, pha4, regulates different gene batteries at different times (Ao et al, 2004;Gaudet and Mango, 2002;Gaudet et al, 2004), and a particular aspect of its multiple capabilities is that the Foxa transcription factor recognizes high and low affinity target sites according to its concentration.…”

A Sea Cucumber Homolog of the Mouse T-Brain-1 is Expressed in the Invaginated Cells of the Early Gastrula in Holothuria leucospilota.

“…4b). Wholemount in situ hybridization using isoform specific probes reveals a complex and dynamic change of expression patterns in the three germ layers (Li et al, 1997;Mitsunaga et al, 1998) (Fig. 5), suggesting that the Otx is not merely required for the differentiation of specific territories.…”