Background Adult echinoderms can completely regenerate major parts of their central

Background Adult echinoderms can completely regenerate major parts of their central nervous system even after severe injuries. death the two most prominent cellular events that take place in the regenerating sea cucumber nervous system shortly after injury. Conclusions In this study we show that overexpression is required for proper dedifferentiation of radial glial cells and for triggering the programmed cell death in the vicinity of the injury. is thus the first transcription factor whose functional role has been experimentally established in echinoderm regeneration. Electronic supplementary material The online version of this article (doi:10.1186/s12861-015-0071-z) contains supplementary material which is available to authorized users. expression showed significant increase in response to the RNC injury. The sea cucumber is a homolog of mammalian Myc proteins global transcription regulators that moderate expression of 10-15 % of the genome and play crucial roles in control of cell growth proliferation balance Neohesperidin between self-renewal and differentiation and apoptosis in various developmental contexts and during oncogenic transformation [11-14]. Interestingly the expression level of the sea cucumber was already elevated during the early response to RNC injury [7]. We therefore hypothesized that this increase in expression was somehow associated with the initiation of CNS regeneration however the specific function(s) of the gene remained unknown. In this study we adapt RNA interference (RNAi)-mediated gene silencing to determine the roles played by during the early post-injury response in the RNC of the brown rock sea cucumber Selenka 1867 We show that elevated levels of during the first two days after the injury are required for proper dedifferentiation of radial glial cells and for initiation of programmed cell death in the vicinity of the lesion. To our knowledge the present study is the first implementation of RNAi methodology in regenerating adult echinoderms. The ability to use functional genomics tools makes it possible at last to experimentally decipher molecular pathways underlying post-traumatic organogenesis in these highly regenerating animals. Results Electroporation of DsiRNAs Neohesperidin reduces Myc expression in the injured radial nerve In order to determine the functional role of in the early response to the CNS injury in transcript (Fig. ?(Fig.2).2). The decision to use longer DsiRNAs with a 25-nt sense strand and a 27-nt antisense strand rather than more traditional shorter 21-nt duplexes was based on the fact that DsiRNAs can be up to 100-fold more efficient than the classical 21-mers [15]. DsiRNAs were injected and electroporated into the RNC (Additional files 1 and 2). Besides reagent delivery the injection procedure also served another function. The diameter of the injection needle was chosen to Mouse monoclonal to BID be greater than the width of the RNC so that complete transection is achieved during the injection procedure (Additional file 2). Two days after surgery quantitative real-time RT-PCR (qRT-PCR) showed that injection and electroporation of either Myc Dsi1 or Myc Dsi2 caused a significant ~1.9-fold decrease in mRNA expression compared with the injection of the control GFP-trageting DsiRNA (Fig. ?(Fig.3a) 3 whereas the animals treated with the control DsiRNA themselves did not Neohesperidin show any significant differences when compared with the animals injected with the vehicle alone. Fig. 2 Diagram showing the sequences of the two transcript. Red and blue letters indicate additional RNA and DNA bases respectively … Fig. 3 RNA interference-mediated knockdown. a expression in the regenerating radial nerve cord on day 2 after injury/DsiRNA injection as determined by qRT-PCR. Two DsiRNA constructs were used Myc Dsi1 and Myc Dsi2 as described in Methods. Expression … Likewise in situ hybridization shows that RNAi-mediated gene targeting causes expression to fall below the detection limit in the cells adjacent to the site of injury/injection. No knockdown occurred when the animals were injected with the control GFP-targeting Dsi RNA (Fig. ?(Fig.33b-c’). Forced downregulation of Myc transcripts impairs dedifferentiation of radial glia Glial dedifferentiation is one Neohesperidin of the key cellular events that take place in the injured sea cucumber RNC shortly after transection [1 3 Under normal conditions fully differentiated radial glial cells show typical palisade morphology with their cell bodies mostly localized to the apical region of the neuroepithelium and the long basal processes extending.