Fig. 5. Concomittant de novo neurogenesis and cnox-2 expression at the early-late stage of head regeneration. (A) Anti-ß-tubulin immunostaining of the nervous system in head-regenerating halves (see also Fig. S3 in the supplementary material). Neuronal processes are absent from the tips (arrowhead, boundary indicated with dots) up to 32 hpa when neuronal precursors differentiate (arrows). (B-G) cnox-2 expression in the presumptive head region (arrows) detected about 20 hours before TBs emerge during both head regeneration at 24 hpa (B,D), and budding at stage 3 (G, arrows). (B) Enlarged views of the head-(arrows) and foot-(arrowhead) regenerating tips depicted in C. Note the absence of cnox-2⁺ cells in the 16 hpa head-regenerating tip. (D-G) cnox-2⁺ cells appear first as pairs of dividing i-cells (D), subsequently as neuronal percursors (E,G) and finally as differentiated neurons (arrows in F) in regenerating tips (D-F) and distal buds (G). (H-J) Transient variations in the cnox-2⁺ cell distribution in regenerating halves (percentages calculated over DAPI-stained ectodermal cells). (H) Decrease in the gastric cnox-2⁺ cell number. (I) Increase in cnox-2⁺ single i-cells at 16 hpa. (J) Early-late increase in cnox-2⁺ cells in head-regenerating tips. Scale bars: 200 µm in C; 100 µm in A,B,G; 20 µm in D,E,G right panel; 10 µm in F. ic, i-cell; ne, neuron; np, neuronal precursor.