Identification of the principal neuropeptide MIP and its action pathway in larval settlement of the echiuran worm Urechis unicinctus

Abstract

Background: Larval settlement and metamorphosis represent critical events in the life history of marine benthic animals. Myoinhibitory peptide (MIP) plays a pivotal role in larval settlement of marine invertebrates. However, the molecular mechanisms of MIP involved in this process are not well understood.

Results: In this study, we evaluated the effects of thirteen MIP mature peptides on triggering the larval settlement of Urechis unicinctus (Xenopneusta, Urechidae), and determined that MIP2 was the principal neuropeptide. Transcriptomic analysis was employed to identify differentially expressed genes (DEGs) between the MIP2-treated larvae and normal early-segmentation larvae. Both cAMP and calcium signaling pathways were enriched in the DEGs of the MIP2-treated larvae, and two neuropeptide receptor genes (Spr, Fmrfar) were up-regulated in the MIP2-treated larvae. The activation of the SPR-cAMP pathway by MIP2 was experimentally validated in HEK293T cells. Furthermore, fourteen cilia-related genes, including Tctex1d2, Cfap45, Ift43, Ift74, Ift22, Cav1 and Mns1, etc. exhibited down-regulated expression in the MIP2-treated larvae. Whole-mount in situ hybridization identified two selected ciliary genes, Tctex1d2 and Cfap45, were specially expressed in circumoral ciliary cells of the early-segmentation larvae. Knocking down Tctex1d2 mRNA levels by in vivo RNA interference significantly increased the larval settlement rate.

Conclusion: Our findings suggest that MIP2 inhibits the function of the cilia-related genes, such as Tctex1d2, through the SPR-cAMP-PKA pathway, thereby inducing larval settlement in U. unicinctus. The study contributes important data to the understanding of neuropeptide regulation in larval settlement.

Keywords: Urechis unicinctus; Cilia-related genes; Gene pathway; Larval settlement; MIP; Neuropeptide.

Fig. 1

MIP-induced larval settlement assays. A-C: Relative height of the larvae treated with 13 MIPs at 1 min, 3 and 5 min; D: Percentage of the larvae in 0–5 cm water layer after treated by 13 MIPs for 5 min, respectively; E: Relative height of the larvae treated by MIP2 at the different concentrations for 5 min. Each rhombus represents a data. All data are represented as the mean ± SEM from three biological replicates. Different letters indicate significant differences (p < 0.05)

Fig. 2

Volcanoplot illustrating the differentially expression of unigenes between the control group larvae and those treated with MIP2 for 3 min. Pink and green spots indicate the differentially expressed unigenes, while blue spots indicate no differentially expression

Fig. 3

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes (DEGs). A: GO enrichment analysis of the down-regulated DEGs; B: GO enrichment analysis of the up-regulated DEGs; C: KEGG enrichment analysis of the down-regulated DEGs; D: KEGG enrichment analysis of the up-regulated DEGs

Fig. 4

CRE-driven luciferase activity in HEK293T cells treated by MIP2. 1: the Forskolin treatment group with 10 µM; 2–5: MIP2 treatment group with 10, 100, 1000, 10,000 nM + 10 µM Forskolin, respectively. Each rhombus represents a data. Different letters indicate significant differences (p < 0.05)

Fig. 5

Relative mRNA level of seven cilia-related genes in the MIP2-treated larvae at different times. Each rhombus represents a data. Different letters indicate significant differences (p < 0.05)

Fig. 6

Settlement rate and target gene mRNA relative levels of the larvae treated with H89. A: Settlement rate of the larvae treated with H89. B: Relative mRNA level of seven ciliary genes in the larvae treated with H89. Data are represented as the mean ± SEM from three biological replicates. Asterisks indicate significant differences between H89 groups and the control group (* p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 7

Location of Tctex1d2 and Cfap45 mRNA in U. unicinctus embryos and larvae detected by Whole-mount in situ hybridization. A: Tctex1d2; B: Cfap45; a and a’: zygote; b and b’: 4-cell embryo; c and c’: 8-cell embryo; d and d’: multicellular embryo; e and e’: blastula; f and f’: gastrula; g and g’: early-trochophore; h and h’: mid-trochophore; i and i’: late-trochophore; j and j’: early-segmentation larva; k and k’: late-segmentation larva; l and l’: worm-shaped larva. Blue indicates the positive signal. AT: apical ciliary tuft; CR: circumoral ciliary ring; TT: telotroch. All scales are 50 μm

Fig. 8

Gene knockdown efficiency and the settlement rate of the larvae after RNAi. A: Gene knockdown efficiency of Tctex1d2 in dsRNA-treated larvae for 72 h. B: Larval settlement rate at 48 h of RNAi. C: Larval settlement rate at 72 h of RNAi. Data are represented as the mean ± SEM from three biological replicates. Asterisks indicate significant differences between Tctex1d2-dsRNA group and blank control group (*** p < 0.001). “ns” indicates no significant differences between EGFP-dsRNA groups and blank control group