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Fig. 4 Figure 4. Template strand segregation in the CMZ of the retina (A) Zygotes were injected with 5.7 pmol F-ara-EdU, followed by incubation in 1 mM F-ara-EdU from 1 to 4 dpf; 10 mM BrdU was administered for 12 h before the end of the chase through incubation. (B) Schematics of the retina highlighting the ciliary marginal zone in red. (C) Models of F-ara-EdU pulse dilution in two hypothetical scenarios: random template strand segregation and nonrandom template strand segregation according to the immortal strand hypothesis. (D) Observed F-ara-EdU label depletion from the CMZ over time. (E) Quantification of F-ara EdU signal decay in CMZ-p (stem cells) vs. INL (postmitotic) cells (see STAR Methods). Statistical significance was determined using the one-sample Wilcoxon test and a hypothetical value of 0.5. n = 70 CMZ-p cells from eight fish total (10–20 cells and two fish per time point). Box and whiskers plot represents median (line in box center), first and third quartile (lower and upper box border, respectively), and minimum and maximum values (whiskers), as well as individual data points (spheres). (F) Examples of F-ara-EdU/BrdU double-positive cell pairs (arrows). (G) F-ara-EdU intensity ratios in daughter cells (n = 21 pairs, see STAR Methods). A binomial curve centered at 0.5 (magenta) was fitted to the data (R2 = 0.98). The blue curve represents the theoretical, normal (Gaussian) distribution for random segregation. Quantification was performed on raw images (see STAR Methods), whereas images shown in (D) and (F) were adjusted in brightness and contrast for better visualization. CMZ, ciliary marginal zone; CMZ-m/c, middle and central peripheral ciliary marginal zone; CMZ-p, peripheral ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; P, postmitotic cells; Ph, photoreceptor layer; SC, stem cells; TA, transit-amplifying cells. ∗p ≤ 0.05, ∗∗∗∗p ≤ 0.0001.