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  • br Material and methods br

    2018-11-08


    Material and methods
    Results
    Discussion The current study is the first report of the dynamic expression of ATF3 by ependymal spinal stem cells, as this protein was localized to the cytoplasm when such cells were quiescent, but was found in their nucleus when cells became activated. This property enabled us to follow up activated ependymal cells that migrated from the central canal toward the ventral and dorsal white matter, forming the FMS that resembles the RMS of the cloprostenol SPCs. The fact that ATF3 immunostaining coincided with well-known SPC markers, such as nestin, vimentin and SOX2, suggests that ATF3 labeling in the spinal cord was primarily expressed by intrinsic stem cells, typically found around the central canal (Hugnot and Franzen, 2011). ATF3 was mainly expressed in the cytoplasm and processes of these cells from P1 onwards, grew during the first postnatal week and remained elevated later. Such ATF3 stained cells in fresh tissue had small diameter, large egg-shaped nucleus, and were closely packed together. Gray matter cells remained, however, devoid of ATF3 staining, adding specificity to the role of this protein for stem cell labeling. Previous reports of the role of ATF3 in the regulation of SPCs are scant. Recent studies (Gao et al., 2013; Gargiulo et al., 2013) have indicated involvement of ATF3 in the control of genes like SOX2 or BMI1, critical for pluripotency and reprogramming of the human embryonic stem cells or glioblastoma stem-like cells. The present observation of ATF3 and SOX2 co-staining is consistent with this possibility. Moreover, the CREB transcription factor family, of which the ATF3 is a member, has an established role in SPC regulation and neurogenesis (Merz et al., 2011; Mantamadiotis et al., 2012). Our results suggest, however, a new role of ATF3 not only in the maintenance of the spinal ependymal cells but also in their activation. When spinal cords were kept in vitro for 24h, we observed a novel phenomenon, namely centrifugal mobilization of ependymal stem cells which formed a migratory chain analogous to the brain RMS (Lois et al., 1996; Wichterle et al., 1997; Tanvig et al., 2009) as they moved away from the central canal to the adjacent ventral and dorsal white matter. When this occurred, ATF3 was clearly expressed in the cell nucleus. This phenomenon developed gradually after 4h and was fully observed at 24h. Interestingly, ependymal cells which did not mobilize, retained their SOX2 nuclear staining, yet lost the ATF3 cytoplasmic one. Hence, ATF3 nuclear labeling could be interpreted as a novel marker of migrating ependymal cells. The fact that in vitro conditions induce mobilization of stem cells has been earlier reported with organotypic brain slices (Tanvig et al., 2009): in such a case RMS toward the olfactory bulb is occurring after several days, while the present report shows a much faster process developing within hours. Our proposal of RMS-like migration is currently based on morphological observations only, since these cells did not stain for NCAM, typical marker of RMS in the olfactory bulb (Hu et al., 1996). Thus, we suggest that the unchanged occurrence of Ki67 or EdU positive cells in FMS at 24h in vitro is compatible with a process of ependymal cell early mobilization rather than proliferation. In support of this hypothesis is the recent observation that, in rat organotypic slices, significant proliferation of neuroprogenitors was detected only after several days in culture (Mazzone et al., 2013). We cannot exclude that ATF3-positive cells might eventually become neurons and/or glia and integrate into spinal networks. This possibility would assume a delayed maturation process outlasting our observation period limited by tissue survival, and actually absent in spinal slice culture (Mazzone et al., 2013). The question then arises regarding the mechanism(s) responsible for activation and mobilization of ependymal cells in vitro, given that spinal networks are fully preserved and viable during this timeframe, including electrophysiological activity of locomotor networks (Taccola et al., 2008). In vivo the ependymal cells are in direct contact with the cerebrospinal fluid (CSF) and numerous blood vessels (Hugnot and Franzen, 2011). Thus, their delayed activation in vitro might be due to the disappearance of yet-unclear signals from the CSF or blood (Menezes et al., 2002), which would normally keep the ependymal cells quiescent (Cheung and Rando, 2013). Additionally, the activating signal might come from a few cells injured during dissection, even if systematic analysis of in vitro tissue showed minimal pyknosis at 24h in vitro (Taccola et al., 2008). A recent review has highlighted how stem cell quiescence is a state maintained by signaling pathways ready to allow rapid activation (Cheung and Rando, 2013). Deciphering the molecular mechanisms regulating stem cell quiescence is, therefore, an important goal for future studies to increase our understanding of tissue regeneration mechanisms in pathological conditions (Cheung and Rando, 2013). Identification of the factors underlying even brain RMS remains incomplete as various chemo-attractants, chemo-repellents, growth factors and cell adhesion molecules have been proposed to guide migrating cells (Menezes et al., 2002; Tanvig et al., 2009). The intracellular presence of other ATF/CREB family members with which ATF3 forms heterodimers may ultimately decide the functional role of ATF3 (Thompson et al., 2009), as much as the influence of regulators like other transcription factors, miRNA or histone modifications (Yun et al., 2010). Future studies are necessary to determine if the FMS pattern of migration detected in vitro appears also in vivo or is due to the experimental tissue preparation with absence of cues from CSF and blood vessels, and if this phenomenon is observed in adult animals as well.