Fig. 1.

Characterization and heterogeneity of proliferating cells in postnatal NP. (a) Confocal microscopy images show immunostaining for Ki67 or EdU (after six-dose administration of EdU) on coronal sections of lumbar and caudal NP (dotted circle) at different ages early after birth. Scale bars, 100 µm. (b) Quantification of the percentages of Ki67⁺ or EdU⁺ NP cells over total NP cells in caudal vertebra (CV) or lumbar vertebra (LV) at each age reveals that sequential EdU administration have higher labeling efficiency than Ki67 immunostaining and caudal NP cells have higher proliferative activity than lumbar NP cells. **P < 0.01, ****P < 0.0001, Two-way ANOVA with Sidak's multiple comparisons test. (c) Volcano plot of differentially expressed genes (DEGs) from RNA-sequencing analysis between caudal NP and lumbar NP displays 994 significantly up-regulated genes and 884 significantly down-regulated genes based on a threshold of fold change ≥ 2 and P < 0.05. Up-regulation of cell cycling genes is indicated by their gene symbols on the plots. (d) KEGG pathway enrichment analysis of DEGs shows that cell cycling pathway is significantly up-regulated in caudal NP compared with lumbar NP. (e) Representative EdU staining images of horizontal sections of mouse disc reveal the location preference of the proliferating NP cells. Boxed area in the left image (scale bars, 100 µm) is shown in higher magnification in the right (scale bars, 20 µm). Arrowheads point EdU⁺ NP cells along the border between the NP and AF indicated by the dotted line. (f) Schema shows the partition of the outer region and inner region divided by the inner circle (defined as the concentric circle with half diameter of NP). (g) Ki67⁺/EdU⁺ NP cells in different regions are counted from both lumbar and caudal IVDs at each age. The percentages of the Ki67⁺/EdU⁺ NP cells in the outer region at different ages indicates that most of the proliferating NP cells are located in the outer region