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BMC Dev Biol
2015 May 30;15:24. doi: 10.1186/s12861-015-0071-z.
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Myc regulates programmed cell death and radial glia dedifferentiation after neural injury in an echinoderm.
Mashanov VS
,
Zueva OR
,
García-Arrarás JE
.
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BACKGROUND: Adult echinoderms can completely regenerate major parts of their central nervous system even after severe injuries. Even though this capacity has long been known, the molecular mechanisms that drive fast and complete regeneration in these animals have remained uninvestigated. The major obstacle for understanding these molecular pathways has been the lack of functional genomic studies on regenerating adult echinoderms.
RESULTS: Here, we employ RNA interference-mediated gene knockdown to characterize the role of Myc during the early (first 48 hours) post-injury response in the radial nerve cord of the sea cucumber Holothuria glaberrima. Our previous experiments identified Myc as the only pluripotency-associated factor, whose expression significantly increased in the wounded CNS. The specific function(s) of this gene, however, remained unknown. Here we demonstrate that knockdown of Myc inhibits dedifferentiation of radial glia and programmed cell 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 Myc overexpression is required for proper dedifferentiation of radial glial cells and for triggering the programmed cell death in the vicinity of the injury. Myc is thus the first transcription factor, whose functional role has been experimentally established in echinoderm regeneration.
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Fig. 1. Organization of the uninjured radial nerve cord and surrounding tissues in the sea cucumber H. glaberrima. Paraffin sections stained with Safranin O, Fast Green and Weigert Iron Hematoxilyn. A Transverse section. B Longitudinal section. bw, body wall; lmb, longitudinal muscle band; rnc, radial nerve cord; tf, tube foot; wvc, water-vascular canal
Fig. 2. Diagram showing the sequences of the two Myc-targeting DsiRNAs (Myc Dsi1 and Myc Dsi2) and their target sites within the open reading frame (ORF) of the H. glaberrima
Myc transcript. Red and blue letters indicate additional RNA and DNA bases, respectively, which distinguish DsiRNAs from classical 21-mer duplexes
Fig. 3. RNA interference-mediated Myc knockdown. a
Myc 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 values are plotted as fold change relative to a negative control (a GFP-targeting DsiRNA) and expressed in a log2 scale. Error bars show standard deviation. ** p<0.01, *** p<0.001. b-c’ Representative in situ hybridization micrographs showing Myc expression in the radial nerve cord on day 2 after transection/DsiRNA injection. The upper (b and b’) and lower (c and c’) rows of micrographs show longitudinal sections of the RNC of an animal treated with a control (GFP-targeting) DsiRNA and an animal injected with one of the Myc-targeting DsiRNAs (Myc Dsi1), respectively. The micrographs on the right (b’ and c’) are high-magnification view of the boxed regions in the main micrographs on the left (b and c, respectively). The red dashed line indicates the position of the plane of injury. Note the absence of in situ hybridization signal from the cell bodies in the apical region of the ectoneural neuroepithelium of the RNC in the animal treated with Myc Dsi1, but not in the animal, which received the control DsiRNA injection
Fig. 4. Representative micrographs showing the effect of Myc knockdown on glial dedifferentiation on day 2 post-injury/DsiRNA injection. The radial glial cells are visualized by immunostaining with the ERG1 monoclonal antibody (red) [17]; the nuclei (in a, b, d, e) were stained with Hoechst (blue). All micrographs are longitudinal sections with the plane of the injury (dashed line) to the right. a and b Control injections of the vehicle (a) and an irrelevant (GFP-targeting) DsiRNA (b). c Higher magnification of the radial nerve cord in a control (vehicle-injected) animal. d and e Injection of Myc-targeting DsiRNAs, Myc Dsi1 (d) and Myc Dsi2 (e). f Higher magnification of the radial nerve cord in a Myc Dsi2-injected animal. Note that in the control animals (a-c) the glial cells loose their long basal processes, while the cell bodies (arrow in c) remain in the apical region of the ectoneural neuroepithelium (en). In contrast, many of the radial glial cells in the animals injected with Myc-targeting DsiRNAs (d-f) retained their basal processes, which extended through the underlying neural parenchyma (asterisk)
Fig. 5. Effect of RNAi-mediated Myc silencing on glial dedifferentiation and programmed cell death. a Relative area occupied by fully differentiated radial glial cells within 1 mm from the wound in control animals injected with the vehicle or an irrelevant (GFP-targeting) DsiRNA (GFP Dsi) and in animals injected with Myc-targeting DsiRNAs (Myc Dsi1 and Myc Dsi2). Day 2 post-injury/DsiRNA injection. b Relative abundance of TUNEL-positive cells in the radial nerve cord within 1mm from the wound on day 2 post injury/DsiRNA injection. The control animals were injected either with the vehicle or with an irrelevant DsiRNA (GFP Dsi). The Myc-targeting DsiRNAs are designated as Myc Dsi1 and Myc Dsi2. The data are plotted as mean ± standard error. * p<0.05, ** p<0.01, *** p<0.001
Fig. 6. Representative micrographs showing the effect of Myc on programmed cell death on day 2 post-injury/DsiRNA injection. The cells undergoing programmed cell death were visualized with TUNEL assay (green). All micrographs are sagittal sections with the plane of the injury (dashed line) to the right. Dotted line indicates the outline of the radial nerve cord. a and b Control injections of the vehicle (a) and irrelevant control GFP-targeting DsiRNA (b). c and d Injection of Myc-targeting DsiRNAs, Myc Dsi1 (c) and Myc Dsi2 (d)
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