Characterization of CMZ cells at single-cell resolution. (A) Schematic illustrating the clonal analysis of CMZ cells. Cre mRNA and H2BPSmOrange mRNA were coinjected into zebrafish embryos (eEF1α:Gal4VP16::UAS:Zebrabow) at one cell stage. Starting at 72 hpf, sparsely labeled cells originating at different CMZ loci were selected and followed for up to 14 dpf. (B) Micrographs illustrating clones derived from the cells at the different loci of the CMZ tip region at 72 hpf (the cells at positions 1–4 are indicated from the left to the right). Gray dashed lines indicate the time window between 3 dpf and 14 dpf. (C) Type I clones originated from the tip-most first or second cell in the CMZ tip and remained in their original loci without cell proliferation as observed until 14 dpf (n = 41). Tracing of one representative Type I clone is shown until 8 dpf. Type II clones consist of marked cells, which originated from cells that were one or two cells further away from the very CMZ tip. These clones remained attached to their original location while expanding continuously (n = 45). One representative type II clone, traced until 5 dpf, is shown. In contrast, Type III clones that originated from cells that were more than three cells away from the CMZ tip at 72 hpf (Type III; n = 87) detached rapidly from the CMZ tip and formed differentiated clones within 1 or 2 d. An example trace of such Type III clone is shown from 3 until 14 dpf. Arrows indicate traced cells. Asterisks indicate CMZ tip cells labeled by H2BPSmOrange but not GFP, GFP, and tdTomato. White dashed lines outline the boundaries of retinas and lenses. Bars, 10 µm.
