| Robert L. Morris, Blair J. Rossetti, Robert T. Manguso, Madeline L. Keyes, Amanda P. Rawson, Tatsushi Shintaku, Ian Greenstein, and Kevin Hewitt |
| The sea urchin embryo has long been a model of choice for studying the formation and function of cilia. In most urchin species, ciliogenesis occurs on all cells just prior to embryo hatching and is regulated thereafter in a tissue-specific manner to generate cilia with different lengths and behaviors. Probing the genome of the purple sea urchin Strongylocentrotus purpuratus using the flagellar proteomes from human and Chlamydomonas, we studied how the urchin regulates tissue-specific differentiation of cilia in development. Genes representing all ciliary protein classes including the flagellar-associated proteins (FAPs) in the Chlamydomonas Flagellar Proteome, were identified in the S. purpuratus genome by reciprocal BLAST to generate a draft sea urchin ciliome. Analysis of S. purpuratus transcriptome data from sequential developmental stages revealed that the majority of putative ciliary genes exhibit four general temporal expression patterns: predominantly zygotic, zygotic/blastular, and blastular (all preceding or coinciding with initial ciliogenesis), and larval (coinciding with formation of ciliary subtypes). Few putative ciliary genes exhibit peak expression during other stages. Some groups of genes whose products presumably function together, such as IFT genes, are expressed prior to or during ciliogenesis but have variable peak expression times. Most FAPs that show modulated expression exhibit a discrete peak at the onset of ciliogenesis in the early blastula. Gene Ontology suggests that ciliary proteins with general metabolic functions exhibit peak expression early in development while many ciliary proteins with more specialized functions exhibit peak expression only after organogenesis. Genes whose products are central to specific processes, such as kinesin-2 family members that drive anterograde IFT, are being targeted by antisense morpholino knockdown to test their roles inferred by expression pattern and Gene Ontology analysis. Using the Chlamydomonas flagellar proteome to develop a draft ciliary proteome in the sea urchin, with its readily available transcriptome data, will allow us to study how the ciliogenic program can be modulated during development to generate cilia with tissue-specific morphologies, motilities, and functions. |