Transcriptome Analysis of Larval Segment Formation and Secondary Loss in the Echiuran Worm Urechis unicinctus

Abstract

The larval segment formation and secondary loss in echiurans is a special phenomenon, which is considered to be one of the important characteristics in the evolutionary relationship between the Echiura and Annelida. To better understand the molecular mechanism of this phenomenon, we revealed the larval transcriptome profile of the echiuran worm Urechis unicinctus using RNA-Seq technology. Twelve cDNA libraries of U. unicinctus larvae, late-trochophore (LT), early-segmentation larva (ES), segmentation larva (SL), and worm-shaped larva (WL) were constructed. Totally 243,381 unigenes were assembled with an average length of 1125 bp and N50 of 1836 bp, and 149,488 unigenes (61.42%) were annotated. We obtained 70,517 differentially expressed genes (DEGs) by pairwise comparison of the larval transcriptome data at different developmental stages and clustered them into 20 gene expression profiles using STEM software. Based on the typical profiles during the larval segment formation and secondary loss, eight signaling pathways were enriched, and five of which, mTOR, PI3K-AKT, TGF-β, MAPK, and Dorso-ventral axis formation signaling pathway, were proposed for the first time to be involved in the segment formation. Furthermore, we identified 119 unigenes related to the segment formation of annelids, arthropods, and chordates, in which 101 genes were identified in Drosophila and annelids. The function of most segment polarity gene homologs (hedgehog, wingless, engrailed, etc.) was conserved in echiurans, annelids, and arthropods based on their expression profiles, while the gap and pair-rule gene homologs were not. Finally, we verified that strong positive signals of Hedgehog were indeed located on the boundary of larval segments using immunofluorescence. Data in this study provide molecular evidence for the understanding of larval segment development in echiurans and may serve as a blueprint for segmented ancestors in future research.

Keywords: Echiura; Hedgehog pathway; Urechis unicinctus; segmentation; transcriptome.

Figure 1

The life cycle of Urechis unicinctus. The gastrula hatches and develops into a swimming larva, the trochophore, in which a prototroch separates the larva into the episphere (upper hemisphere) and the hyposphere (lower hemisphere) to form the head and the trunk, respectively. The segments occur firstly in the early-segmentation larva and then maintain through the late-segmentation larva. The secondary loss of the segments appears in the worm-shaped larva whose inhabit mode changes from free-living into burrowing in the sediment. A body plan without segments is kept in adult U. unicinctus.

Figure 2

Species distribution of the unigenes from U. unicinctus larval transcriptome with blast in NR database.

Figure 3

DEGs profiles of U. unicinctus larval transcriptome with a pairwise comparison of each time point.

Figure 4

Expression patterns of the DEGs across LT, ES, SL, and WL stages of U. unicinctus. The x-axis represents the larval developmental stages, which are LT, ES, SL, and WL from left to right in each profile; the y-axis represents DEGs expression trends.

Figure 5

Validation of the RNA-Seq data using qRT-PCR. The blue columns represent the qRT-PCR results; the red lines show the FPKM value. Different characters indicate the significant difference (p < 0.05), with the uppercase letter for qRT-PCR and the lowercase for RNA-Seq. smo belongs to profile11, wnt11 and post2 belong to profile18, nk1 belongs to profile16, abca3 belongs to profile10, nlk and odd belong to profile9 and rp-sae belongs to profile2, respectively.

Figure 6

Hierarchical clustering of the segment formation homologous genes in U. unicinctus larval transcriptome. (A), Gap genes and pair-rule genes; (B), segment polarity genes; (C), homeotic genes. The clustering indicates similar expression patterns among the genes. The expression levels of genes are presented in the color tape from low (green) to high (red).

Figure 7

Immunofluorescence assay of HH in U. unicinctus larvae during the segment formation and secondary loss. (A–D) correspond to LT, ES, SL, and WL of U. unicinctus, respectively. (A–C), ventral views; (D), lateral view.