Cooption of regulatory modules for tektin paralogs during ciliary band formation in a marine annelid larva.

Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.

Genes with spiralian-specific protein motifs are expressed in spiralian ciliary bands.

Spiralia is a large, ancient and diverse clade of animals, with a conserved early developmental program but diverse larval and adult morphologies. One trait shared by many spiralians is the presence of ciliary bands used for locomotion and feeding. To learn more about spiralian-specific traits we have examined the expression of 20 genes with protein motifs that are strongly conserved within the Spiralia, but not detectable outside of it. Here, we show that two of these are specifically expressed in the main ciliary band of the mollusc Tritia (also known as Ilyanassa). Their expression patterns in representative species from five more spiralian phyla-the annelids, nemerteans, phoronids, brachiopods and rotifers-show that at least one of these, lophotrochin, has a conserved and specific role in particular ciliated structures, most consistently in ciliary bands. These results highlight the potential importance of lineage-specific genes or protein motifs for understanding traits shared across ancient lineages.

Taxon-specific expansion and loss of tektins inform metazoan ciliary diversity.

BACKGROUND: Cilia and flagella are complex cellular structures thought to have first evolved in a last ciliated eukaryotic ancestor due to the conserved 9 + 2 microtubule doublet structure of the axoneme and associated proteins. The Tektin family of coiled-coil domain containing proteins was previously identified in cilia of organisms as diverse as green algae and sea urchin. While studies have shown that some Tektins are necessary for ciliary function, there has been no comprehensive phylogenetic survey of tektin genes. To fill this gap, we sampled tektin sequences broadly among metazoan and unicellular lineages in order to determine how the tektin gene complements evolved in over 100 different extant species. RESULTS: Using Bayesian and Maximum Likelihood analyses, we have ascertained with high confidence that all metazoan tektins arose from a single ancestral tektin gene in the last common ancestor of metazoans and choanoflagellates. Gene duplications gave rise to two tektin genes in the metazoan ancestor, and a subsequent expansion to three and four tektin genes in early bilaterian ancestors. While all four tektin genes remained highly conserved in most deuterostome and spiralian species surveyed, most tektin genes in ecdysozoans are highly derived with extensive gene loss in several lineages including nematodes and some crustaceans. In addition, while tektin-1, - 2, and - 4 have remained as single copy genes in most lineages, tektin-3/5 has been duplicated independently several times, notably at the base of the spiralian, vertebrate and hymenopteran (Ecdysozoa) clades. CONCLUSIONS: We provide a solid description of tektin evolution supporting one, two, three, and four ancestral tektin genes in a holozoan, metazoan, bilaterian, and nephrozoan ancestor, respectively. The isolated presence of tektin in a cryptophyte and a chlorophyte branch invokes events of horizontal gene transfer, and that the last common ciliated eukaryotic ancestor lacked a tektin gene. Reconstructing the evolutionary history of the tektin complement in each extant metazoan species enabled us to pinpoint lineage specific expansions and losses. Our analysis will help to direct future studies on Tektin function, and how gain and loss of tektin genes might have contributed to the evolution of various types of cilia and flagella.

A transcriptional blueprint for a spiral-cleaving embryo.

BACKGROUND: The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RESULTS: RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. CONCLUSIONS: Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

Structure, phylogeny, and expression of the frizzled-related gene family in the lophotrochozoan annelid Platynereis dumerilii.

BACKGROUND: Wnt signaling pathways are highly conserved signal transduction pathways important for axis formation, cell fate specification, and organogenesis throughout metazoan development. Within the various Wnt pathways, the frizzled transmembrane receptors (Fzs) and secreted frizzled-related proteins (sFRPs) play central roles in receiving and antagonizing Wnt signals, respectively. Despite their importance, very little is known about the frizzled-related gene family (fzs & sfrps) in lophotrochozoans, especially during early stages of spiralian development. Here we ascertain the frizzled-related gene complement in six lophotrochozoan species, and determine their spatial and temporal expression pattern during early embryogenesis and larval stages of the marine annelid Platynereis dumerilii. RESULTS: Phylogenetic analyses confirm conserved homologs for four frizzled receptors (Fz1/2/7, Fz4, Fz5/8, Fz9/10) and sFRP1/2/5 in five of six lophotrochozoan species. The sfrp3/4 gene is conserved in one, divergent in two, and evidently lost in three lophotrochozoan species. Three novel fz-related genes (fzCRD1-3) are unique to Platynereis. Transcriptional profiling and in situ hybridization identified high maternal expression of fz1/2/7, expression of fz9/10 and fz1/2/7 within animal and dorsal cell lineages after the 32-cell stage, localization of fz5/8, sfrp1/2/5, and fzCRD-1 to animal-pole cell lineages after the 80-cell stage, and no expression for fz4, sfrp3/4, and fzCRD-2, and -3 in early Platynereis embryos. In later larval stages, all frizzled-related genes are expressed in distinct patterns preferentially in the anterior hemisphere and less in the developing trunk. CONCLUSIONS: Lophotrochozoans have retained a generally conserved ancestral bilaterian frizzled-related gene complement (four Fzs and two sFRPs). Maternal expression of fz1/2/7, and animal lineage-specific expression of fz5/8 and sfrp1/2/5 in early embryos of Platynereis suggest evolutionary conserved roles of these genes to perform Wnt pathway functions during early cleavage stages, and the early establishment of a Wnt inhibitory center at the animal pole, respectively. Numerous frizzled receptor-expressing cells and embryonic territories were identified that might indicate competence to receive Wnt signals during annelid development. An anterior bias for frizzled-related gene expression in embryos and larvae might point to a polarity of Wnt patterning systems along the anterior-posterior axis of this annelid.

Expression of the wnt gene complement in a spiral-cleaving embryo and trochophore larva.

The highly conserved wnt gene family has roles in developmental processes ranging from axis formation to cell fate determination. The polychaete Platynereis dumerilii has retained 12 of the 13 ancient wnt subfamilies and is a good model system to study the roles of the wnt ligands in spiralian development. While it has been shown that Platynereis uses a global beta-catenin-mediated binary cell fate specification module in development, the early roles of the 12 wnt genes present in Platynereis are unknown. Transcriptional profiling by RNA-Seq during early development and whole-mount in situ hybridization of embryo and larval stages were used to determine the temporal and spatial regulation of the wnt complement in Platynereis. None of the 12 wnt transcripts were maternally provided at significant levels. In pregastrula embryos, zygotic wntA, wnt4, and wnt5 transcripts exhibited distinctive patterns of differential gene expression. In contrast, in trochophore larvae, all 12 wnt ligands were expressed and each had a distinct expression pattern. While three wnt ligands were expressed in early development, none were expressed in the right place for a widespread role in beta-catenin-mediated binary specification in early Platynereis development. However, the expression patterns of the wnt ligands suggest the presence of numerous wnt signaling centers, with the most prominent being a bias for staggered posterior wnt expression in trochophore larvae. The similarity to wnt expression domains in cnidarians around the blastopore and the tail organizer in chordates supports a hypothesis of a common evolutionary origin of posterior organizing centers.