(A) Baseline proliferation in AxFUCCI spinal cord is not significantly different from d/d controls. EdU was administered in a 2 hours pulse to uninjured AxFUCCI or d/d control animals. Tails were harvested and sectioned to quantify EdU-labeled proliferative cells in the spinal cord. ns: not significant, p=0.62 (two-sample t-test). Error bars indicate standard deviation. n = 5 tails (control) or three tails (AxFUCCI), 750 cells counted in total for each, of which ~50 cells were EdU-labeled. (B) AxFUCCI tails regenerate with normal kinetics. Spinal cord outgrowth was measured every day up to 5 days after AxFUCCI tail amputation and plotted together with measurements previously obtained from d/d control animals (Rodrigo Albors et al., 2015). See Section 2.11 for details of how spinal cord outgrowth was measured. (C) DNA quantification by flow cytometry. Relative DNA content in G0/G1-AxFUCCI (green), S/G2-AxFUCCI (magenta), or Transition-AxFUCCI (gray) cells dissociated and harvested from uninjured AxFUCCI axolotl tails. Cells were incubated with Hoechst DNA stain for 60 min prior to analysis. (D) DNA quantification from AxFUCCI tissue sections. Single-section confocal image of a 10 μm-thick AxFUCCI spinal cord cross-section, co-stained with DAPI (gray) to label DNA. The DNA intensity of G0/G1-AxFUCCI cells (green) was compared to that of S/G2-AxFUCCI cells (magenta). The center panel depicts the cells that satisfy the criteria for quantification – they must span the spinal cord, contact the lumen, and not be obscured by neighboring cells. The AxFUCCI animals were also pulsed with EdU for 8 hours prior to tail harvesting. S/G2-AxFUCCI cells, but not G0/G1-AxFUCCI cells, incorporated EdU (yellow, right panel). (E) Histogram depicting the DAPI intensities of AxFUCCI cells quantified in (D). The mean intensity of G0/G1-AxFUCCI cells was set to an arbitrary value of 1 (2n). S/G2-AxFUCCI cells have a significantly higher DNA content than G0/G1-AxFUCCI cells (P = 2.20 × 10–16, Welch’s test). n = 3 tails (a total of 341 G0/G1-AxFUCCI cells vs. 157 S/G2-AxFUCCI cells). (F) Correlation of EdU labeling (see panel D) with each AxFUCCI-expressing population. n = 3 tails, 819 AxFUCCI-expressing cells were counted in total, of which 171 had incorporated EdU. (G) Spinal cord neurons express G0/G1-AxFUCCI. NeuN-expressing neurons are located on the periphery of the spinal cord. 98% ± 1.5% of neurons expressed G0/G1-AxFUCCI, and the remainder did not express any AxFUCCI. n = 5 tails, a total of 2,170 NeuN+ cells counted. (H) Ependymal cells express all AxFUCCI combinations. Sox2-expressing ependymal cells are located on the interior of the spinal cord, lining the lumen.