Evolution of the chordate regeneration blastema: Differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona

Abstract

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration.

A microRNA-mRNA expression network during oral siphon regeneration in Ciona

“…This study provides a resource that will facilitate future investigation into the genetic requirements of appendage regeneration in chordates. To make our results comparable with those of a previous microarray study (Hamada et al, 2015) we also sequenced RNA from non-regenerating OSs and identified several of the same genes that they identified as the most significantly differentially expressed at 3 days postamputation.…”

“…A microarray-based study of gene expression during OS regeneration was recently published (Hamada et al, 2015). This study reported the 30 genes with the largest changes relative to NR OS at 3, 6 and 9 D. We compared the list of genes reported as DE at day 3 of regeneration with an analogous list derived in this study (Table S4).…”

“…These time points were chosen to represent three stages of regeneration observed in vertebrates: wound healing, transition and redevelopment. Previous reports of differential expression had used NR OSs for comparison (Hamada et al, 2015). It was expected that comparisons of subsequent stages with either D0 or NR OSs would generate different categories as being enriched, and that by using both a fuller picture would be generated.…”

“…The ability to detect transcript level changes in a comprehensive and unbiased manner can identify previously unknown coordination between signaling pathways, transcription factors and effector genes required for regeneration. There have been several transcriptome-wide studies of gene expression of both mRNA (Campbell et al, 2011;Hamada et al, 2015;Knapp et al, 2013;Love et al, 2011;Monaghan et al, 2009;Schebesta et al, 2006;Stewart et al, 2013;Wu et al, 2013) and microRNA (miRNA) (Gearhart et al, 2015;Holman et al, 2012;Hutchins et al, 2016;Thatcher et al, 2008) during appendage regeneration. However, none of these studies connected the expression profiles of miRNA to those of mRNA.…”

Integrin-alpha-6+ Stem Cells (ISCs) are responsible for whole body regeneration in an invertebrate chordate

“…We next aimed to analyze the expression of components of the Notch and Wnt signaling pathways in proliferating cells from the blood during the early time points of WBR. These pathways are known to play roles in regulating stem cell activity, proliferation of blastema cells and cellular differentiation in other organisms [31][32][33][34][35][36][37][38] and are upregulated during WBR in Botrylloides [39]. We isolated cycling G2/M cells by flow cytometry from stage 0 as well as at 24h and 48h after injury of vessel fragments.…”