Basal tubercle cells display increased proliferation rates, upper cells increased levels of activated caspase 3 in the absence of other hallmarks of cell death.

(A–C) TEM images of same specimen as shown in Figure 3, revealing nuclei with normal chromatin in basal and spinous cells of breading tubercles (A,B), whereas the chromatin of outer cap cells is more condensed (C). (D,E) TUNEL staining of pectoral fins of male fish; 1 year of age; whole mount (D) and transverse sections (E). Single TUNEL-positive cells are present in tubercle-free, inter-ray region (D, arrows) and in basal layer of breeding tubercles (E; arrow), whereas upper layers of breeding tubercles are TUNEL-negative (E). Inset in (E) shows a section through the inter-ray region after DNase treatment, with TUNEL signals in all nuclei (positive control). (F–I) anti-activated caspase 3 immunofluorescence (IF) (F), anti-p63 IF (G), anti-BrdU iIF after BrdU incorporation for 12 hours (H,I), co-stained with DAPI; transverse section through breeding tubercle of lower jaw; 1 year old fish (F–H); 31 day old fish (I). Arrows point to regular epidermis. (J) Percentages of BrdU-positive nuclei of specimens as shown in (H,I) in regular epidermis (red) and lower (BrdU-positive) layers of breeding tubercles (blue). Standard deviations and numbers of evaluated samples (n) are indicated. For all stages, differences between regular epidermis and breeding tubercles are statistically significant: 27 d, p = 0.000431; 31 d, p = 0.00000006; 35 d, p = 0.000178; 1 yr, p = 0.000295. Abbreviations: d, days; yr, year.