?(Fig.3j).3j). to compare regenerative and non-regenerative responses in the same specie [5, 40]. Pre-metamorphosis stages (NF stage 48C54) show a very efficient SC regeneration and are considered regenerative stages (R-stage). This ability is lost during metamorphosis (NF stage 66), and post-metamorphic animals including froglets are unable to regenerate the SC therefore are denominated as non-regenerative stages (NR-stages) [41C47]. At R-stages, most cells lining the CC have a radial glial morphology, are uniciliated, and express Sox2 [48]. While in NR-Stages, most cells lining the CC are multiciliated with an advance maturation and differentiation α-Terpineol state and only few cells are uniciliated [48]. In R-stages, but not in the NR-stages, SCI induces a massive and transient proliferation of Sox2/3+ progenitor that is required for proper spinal cord regeneration, and formation of new neurons [46, 47]. In R-stages, glial cells closely associated with growing axons in the ablation gap, suggesting a possible role for them in generating a glial bridge to aid in axonal regeneration [42]. Little evidence of glial scar formation in non-regenerative stages of has been reported, so far, α-Terpineol scar tissue was found encapsulating the α-Terpineol end of the spinal cord lesion in post-metamorphic frogs [44]. Here, we compare the cellular response to SCI of the SC central canal, between the R- and NR-stages of regulatory regions to drive EGFP expression. Characterization of this transgenic line showed expression in radial glial cells in R-stages, and astrocytes in NR-stage froglets. RNAseq analysis of the cells expressing the transgene in R-stage, demonstrated that they correspond to NSPCs. At the R-stage spinal cord, injury activates proliferation of NSPCs that differentiate into α-Terpineol neurons. Ablation of these cells abolishes proper regeneration, confirming that are necessary for a functional regeneration of the spinal cord at NF stage Rabbit polyclonal to PRKCH 50. Results Cellular response to injury in regenerative and non-regenerative stages The cellular organization of the spinal cord CC in changes between regenerative and non-regenerative stages [48]. To determine the cellular response to spinal cord injury between regenerative (R-stages, NF stage 50) and non-regenerative (NR-stages, NF stage 66) stageswe performed a detailed cellular analysis. The spinal cord of R-stage animals was injured by full transection as described previously [40] (Fig.?1a), and tissues were analyzed by light and electron microscopy at different days post transection (dpt). At 2 dpt (Fig. ?(Fig.1b,1b, d), a complete sealing of the rostral stump was observed (Fig. ?(Fig.1b,1b, arrowheads in Fig. ?Fig.1d).1d). The cells lining the CC close to the injury site were not affected by α-Terpineol the lesion. To identify ultrastructural changes in CC cells after SCI, we analyzed ultrathin sections. Cells lining the CC, characterized in the control as type I, II or III [48], lack junction complexes compared to controls (Fig. ?(Fig.1e,1e, arrowheads), contain swelled mitochondria in their apical pole (Fig. ?(Fig.1e,1e, arrow), and frequent centriolar satellites were found (see supplementary material, Fig. S1A, arrowheads). As expected, we identified abundant cells showing mitotic figures (27,75 mitotic cells/m2 ?105, sd. 4,32) indicating cell division [46, 47]. Almost half of the cellular clusters undergoing cell division have no contact (12 mitotic cells/m2 ?105, sd. 2,55) with the lumen of the central canal (Fig. ?(Fig.1f),1f), while the other half (15,75 mitotic cells/m2 ?105, sd. 2,59) are in direct contact with it (Fig. ?(Fig.1g).1g). Although in a lower proportion, cell division in the CC has been also observed in uninjured animals [47, 48]. Conspicuous among the cells lining the CC was the presence of donut-.