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.

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

“…Data from U. unicinctus larval transcriptomes [59,60] and adult transcriptome [61] were used in this study.…”

“…The frequency of each amino acid in the alignment result was presented using the online tool WebLogo [73]. The hierarchical clustering of the pNP genes according to their FPKM values in the U. unicinctus larval transcriptome [59] was performed by an online tool (https://www.omicshare.com/tools/). [74], and expression ratios were calculated using the 2 -ΔΔCt method.…”

“…and Urechis-speci c. Previously established metazoan neuropeptide families are indicated in red [28]. (B) Hierarchical clustering of the neuropeptide precursor genes in U. unicinctus larval transcriptomes [59]. LT, late-trochophore (25 dpf, pelagic larva); ES, earlysegmented larva (32 dpf, pelagic larva); SL, segmented larva (35 dpf, competent larva); WL, worm-shaped larva (42 dpf, benthic larva).…”

Identification of the Neuropeptide Precursor Genes Potentially Involved in the Larval Settlement in the Echiuran Worm Urechis unicinctus

“…Data from U. unicinctus larval transcriptomes [59,60] and adult transcriptome [61] were used in this study. To search for transcripts encoding putative neuropeptides or peptide hormone precursor proteins in U. unicinctus, the homologous sequences previously identified in annelids (Platynereis dumerilii [29], Capitella capitate [43] and Helobdella robusta [43]), molluscs (Patinopecten yessoensis [44], Pinctata fucata [62], Lottia gigantea [63], Crassostrea gigas [62] and Deroceras reticulatum [64]) and echinoderms (Asterias rubens [65], Ophionotus Victoria [66], Strongylocentrotus purpuratus [67] and Apostichopus japonicus [68]) were downloaded from NCBI and used as queries in tBLASTn searches of the assembled U. unicinctus transcriptome database using BioEdit v7.0.9 with an E-value cutoff of 1e-5.…”

Muscular Development in Urechis unicinctus (Echiura, Annelida)

“…The Urechis genus may hold important clues to the genetic basis of evolutionary gain and loss of segmentation, due to its nested position within the Annelida (i.e., sister to capitellid polychaetes) [10,11]. Recently, the transcriptome analysis during the development of U. unicinctus has been reported [12,13]. However, little is known about the molecular basis of muscular development in U. unicinctus.…”