Spec3: embryonic expression of a sea urchin gene whose product is involved in ectodermal ciliogenesis.

Abstract

We have characterized the temporal and spatial expression of Spec3 mRNA in embryos of the sea urchin, Strongylocentrotus purpuratus. This mRNA, 2.0 kb in length, is present at low levels in unfertilized eggs but accumulates rapidly during cleavage, increasing 50-fold by hatching blastula stage. Message levels then decline abruptly, remain constant during mesenchyme blastula and gastrula stages, and increase again during prism and pluteus stages. This accumulation pattern is quite similar to that of the ectodermally expressed 13-tubulin mRNAs described recently by Harlow and Nemer (1987a). In situ hybridization shows that although Spec3 message accumulates in all blastomeres at early blastula stages, it later becomes restricted to ectoderm. By late blastula stage, hybridization is strongest in the animal hemisphere. At gastrula, signals are variable over ectoderm, and by pluteus, grains are concentrated in the ciliary band, though present in other ectodermal cells as well. Deciliation and regeneration of cilia in gastrula-stage embryos results in a four-to fivefold increase in Spec3 mRNA levels, implying that the Spec3 gene product is associated with ciliogenesis. Spec3 mRNA is encoded by a single gene in the haploid genome, and characterization of the gene shows that it contains three exons that encode an open reading frame for a hydrophobic protein of 21.6 kD. The reading frame reveals that the carboxy-terminal part of the protein contains two long hydrophobic stretches, 31 and 37 residues long, separated by short hydrophi!ic regions of six to eight residues. The presence of these two distinct hydrophobic stretches suggests that the Spec3 protein contains two ~-helical domains that either span the lipid bilayer or are associated with some other hydrophobic environment.

Localization of the sea urchin Spec3 protein to cilia and Golgi complexes of embryonic ectoderm cells.

“…Golgi complexes and cilia of oral and aboral ectoderm cells were stained. Brightly stained apical structures or cilia were never observed in endoderm cells (which are ciliated) or mesenchyme cells, consistent with the great enrichment of Spec3 mRNA in the ectoderm (Eldon et al 1987).…”

“…Acidic and basic amino acids are rare (only 12 of 208 amino acids), with 11 clustered in short hydrophilic regions bounding two long hydrophobic domains of nonpolar amino acids in the carboxy-terminal half of the protein ( Fig. 1; Eldon et al 1987). The proline content of the protein (14%) is unusually high, and the proline distribution asymmetrical.…”

“…In a previous study, we characterized a gene, Spec3, whose expression is associated with the process of ectodermal ciliogenesis in Strongylocentrotus purpuratus (Eldon et al 1987). The Spec3 message displays a temporal and spatial pattern of accumulation expected for a message whose translation product is involved in ciliogenesis.…”

“…The Spec3 protein has not been identified in embryos, but the reading frame of the Spec3 mRNA encodes a 21,600 dalton protein of unusual hydrophobicity and high proline content, lacking substantial sequence similarities to any known proteins (Eldon et al 1987). To demonstrate the relationship between the Spec3 protein and cilia, we generated an antibody against the amino terminus of the Spec3 reading frame.…”

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

“…It may be relevant that Spec3, an ectoderm-specific protein whose synthesis correlates with ciliogenesis, appears first in the Golgi complex before incorporation into motile cilia, but prevention of its translocation to the axoneme does not impair motility (Eldon et al, 1987(Eldon et al, , 1990. Although its N-terminal region is exposed at the ciliary membrane surface, the protein remains anchored to the axoneme after detergent extraction, suggesting that it may be part of a transmembrane-axonemal complex that is not part of the motile apparatus per se but may be involved in protein translocation.…”