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Fig. 4 Proliferative tissue degeneration is accompanied by a sustained decrease in proliferation, acute apoptotic responses, and progressive accumulation of DDR foci.
Representative immunofluorescence images of tissue sections in F1 tert-/- and tert+/+ zebrafish show levels of proliferation (PCNA), apoptosis (TUNEL), DNA damage (53BP1) and senescence-associated β–galactosidase at the ages of 3 to c.12 months. Proliferative tissues such as A) testes, B) head kidney and C) gut sections show sustained significant decrease in proliferation in tert-/- as compared to tert+/+ siblings (panels b, d and e) (p<0.001) and an acute apoptotic response at 3 months of age (p<0.001), which clears by c. 12 months (panels g, i and j). This is accompanied by a progressive increase in 53BP1 foci, reaching maximum significance at c. 12 months (panels l, n and o; p<0.001). This coincides with the presence of senescence-associated β –galactosidase at c.12 months (panels s and t). Note in the testes that most of apoptosis (TUNEL) seems to localize to the spermatogenic zone (Ag, dashed outline) and panel E, where we see an increase in TUNEL-labelled germ cells, labelled with the specific marker PLZF. Most of DNA damage (53BP1) locates to the proliferative zone of maturing spermatocytes (Al, uniform outline). Note in the head kidney B), both the proliferative haematopoietic tissue (Ba–d, arrows) and the non-proliferative mesonephric tubule epithelium (Ba–d, dashed outline) are affected by increased apoptosis (Bg, I, j), DNA damage (Bl, n and o) and senescence (Bs and t) in the tert-/-. D) Muscle, a largely non-proliferative tissue (Da–d) shows significant accumulation of DNA damage foci at in tert-/- by the age of c.12 months (Dn and o; p<0.001), when the muscle fibres are already atrophic (Dn, dashed outline). Quantifications were performed in at least 3 different fields of view of at least 3 different individuals of each genotype at the different time-points indicated in the graphs. Gut IF quantifications were calculated as number of positive cells per “crypt” zone (C) uniform square outline exemplified). Other tissues′ IF was quantified as overall % positive cells. β-galactosidase was quantified as % area stained blue, per field of view. Data are represented as mean +/- SEM. Scale bar = 50 ;mu;m.