Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus

Abstract

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb’dome), the Janus kinase Hopscotch (Gb’hop) and the transcription factor STAT (Gb’Stat) were cloned, and RNAi was performed against these genes. Gb’domeRNAi, Gb’hopRNAi and Gb’StatRNAi crickets showed defects in leg regeneration. Blastema expression of Gb’cyclinE was decreased in the Gb’StatRNAi cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb’fatRNAi or Gb’wartsRNAi was suppressed by RNAi against Gb’Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Regenerated leg segment patterns are regulated epigenetically by histone H3K27 methylation in the cricket Gryllus bimaculatus

“…During the dedifferentiation and redifferentiation processes, the expression profiles of differentiated cell-related and stem cell-related genes change epigenetically (Barrero and Izpisua Belmonte, 2011;Katsuyama and Paro, 2011;Tamura et al, 2010). Our previous study showed that the expression levels of the epigenetic modifiers Gb'E(z) and Gb'Utx are upregulated during cricket leg regeneration (Bando et al, 2013). In this study, we examined the roles of these factors in leg regeneration.…”

“…We previously showed that Gb'E(z) and Gb'Utx expression is upregulated within 24 h after cricket leg amputation (Bando et al, 2013), suggesting that Gb'E(z) and Gb'Utx might be involved early in the regeneration process, such as during the dedifferentiation into blastema cells. During Drosophila imaginal disc regeneration, the expression of Polycomb group genes, including E(z), is directly downregulated by JNK signalling in the wounding edge, increasing the plasticity of differentiated cells for promoting blastema formation (Lee et al, 2005).…”

“…The highest RPKM (reads per kilobase per million reads) ratio observed between the blastema and non-regenerative tissue was 8.9 for Gb'Utx, which encodes a histone H3K27 demethylase. Gb'E(z), which encodes a histone H3K27 methyltransferase, and Gb'Polycomb (Gb'Pc), which encodes a histone H3K27me3-binding protein, were also upregulated in the blastema (Bando et al, 2013).…”

“…The Wg/Wnt and Jak/STAT signalling pathways are essential for blastema cell formation because RNAi silencing of these signalling pathways causes regeneration defects (Bando et al, 2013;Nakamura et al, 2007). If Gb'E(z) and Gb'Utx are essential for the dedifferentiation process to form the blastema, RNAi against Gb'E(z) and Gb'Utx should cause complete regeneration defects.…”

“…In a previous study to identify the molecules that undergo expression changes in the blastema, we performed a comparative transcriptome analysis and found that the expression of several epigenetic modifiers is upregulated in the blastema (Bando et al, 2013). In the present study, we focused on the function of the Gryllus homologues of Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx).…”