Conserved Signaling Pathways in the Ciona robusta Gut

Abstract

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

Keywords: Ciona robusta; RNAseq; digestive tract; gut; signaling pathway; urochordate.

Figure 1

RNA-seq approach and preliminary results. (A) The anatomical parts of the Ciona gastrointestinal tract analyzed separately. S = stomach, PI = proximal intestine, DI = distal intestine, blue. (B) Principal component analysis, constructed based on log-transformed Transcript Per Million (TPM) expression values of all genes, summarizing the relatedness of the transcriptional profiles of the three biological samples of S (SA, SB, and SH), PI (PIA, PIB, and PIH), and DI (DIA, DIB, and DIH). (C) Heat map representing the log-transformed TPM expression levels of all DEGs characterized by fold change (FC) > 10 in at least one pairwise comparison among S, PI, and ID. DEGs were hierarchically clustered based on Euclidean distance, using an average linkage method. (D) Heat map representing the log-transformed TPM expression levels of the Hox and paraHox genes, hierarchically clustered based on Euclidean distance, using an average linkage method. Genes displaying statistically significant over-expression in one of the three GI tracts are boxed. The terms A, B, and H refer to different individual animals; for details, see the Material and Methods (Section 4).

Figure 2

Go enrichment and validation of digestion-related genes. Dendrogram representation of the significantly enriched GO terms in the S vs. I pairwise comparison. (A) GO terms associated with DEGs upregulated in S. (B) GO terms associated with DEGs upregulated in I. The size of the GO terms indicates the observed/expected ratio, whereas the color scale represents the statistical significance. GO term clustering was based on the presence of shared DEGs. (C) Validation by real-time quantitative PCR (RT-qPCR) of the digestion-related genes. Violin plots represent relative expression calculated using the ΔCt method, normalization against ELF1. N = 4. ns: not significant, ** p < 0.01 *** p < 0.001, **** p < 0.0001.

Figure 3

Canonical WNT signaling reconstruction. (A) Schematic view of the WNT/βcatenin pathway realized using BioRender.com (accessed on 13 September 2023). (B) STRING networks represent the interactions among selected genes linked with the Wnt pathway, arbitrarily classified as ligands, receptors, nuclear effectors, modulators, targets, and related genes. Only mouse genes sharing at least one orthologous gene with C. robusta are shown. Gene interaction networks were constructed based on known or inferred relationships among mouse orthologs. (C) Heat map representing the log-transformed TPM expression levels of the main genes (excluding target and related genes) included in the STRING network, observed in C. robusta S, PI, and DI samples.

Figure 4

Canonical Notch signaling reconstruction. (A) Schematic view of the Notch pathway realized using BioRender.com (accessed on 13 September 2023). (B) STRING networks representing the interactions among selected genes linked with the Notch pathway, arbitrarily classified as ligands, receptors, nuclear effectors, modulators, targets, and related genes. Only mouse genes sharing at least one orthologous gene with C. robusta are shown. Gene interaction networks were constructed based on known or inferred relationships among mouse orthologs. (C) Heat map representing the log-transformed TPM expression levels of the main genes (excluding target and related genes) included in the STRING network observed in C. robusta S, PI, and DI samples.

Figure 5

Canonical TGFβ-BMP signaling reconstruction. (A) Schematic view of the TGFβ-BMP pathway realized using BioRender.com (accessed on 13 September 2023). (B) STRING networks representing the interactions among selected genes linked with the TGFβ-BMP pathway, arbitrarily classified as ligands, receptors, nuclear effectors, modulators, targets, and related genes. Only mouse genes sharing at least one orthologous gene with C. robusta are shown. Gene interaction networks were constructed based on known or inferred relationships among mouse orthologs. (C) Heat map representing the log-transformed TPM expression levels of the main genes (excluding target and related genes) included in the STRING network, observed in C. robusta S, PI, and DI samples.