Reduced neoblast colony size following lineage specification block.
(A) Models for lineage growth. Potential outcomes of lineage inhibitions were assessed by altering the colony growth and degradation parameters. Applying known lineage frequencies (Raz et al., 2021) to the models indicated that inhibiting production of a major lineage could dramatically impact colony size (left, epidermis). By contrast, inhibition of smaller lineages (middle, right) might have a smaller impact, which would be difficult to detect (Blue: increase in symmetric renewal; red: unchanged renewal and degradation; yellow: increase neoblast degradation). Growth was calculated using the following values: N0 = 5, τ = 29.7 hr, and growth lag time of 5 days (Raz et al., 2021; Wagner et al., 2012). (B) Shown are counts (left) of neoblasts in colonies at three time points following irradiation, which was followed by inhibition of zfp-1 or control by RNAi. Lineage inhibition resulted in a highly significant decrease in colony size at later time points (box indicates interquartile range (IQR), whiskers show range, bar indicates the median). Number of control colonies analyzed at 7 dpi n = 28; at 9 dpi n = 30; at 12 dpi n = 30; Number of zfp-1 (RNAi) colonies analyzed at 7 dpi n=15; at 9 dpi n=30; at 12 dpi n = 30. Representative colonies (z-projection) are shown (right). (C) Comparison of absolute (top-left) or normalized (top-right) H3P+ cell number in zfp-1 (RNAi) and control colonies showed a non-significant difference (Methods). Representative H3P labeling images are shown (Bottom). Importantly, the number of detectable H3P+ cells was small. (D) Comparison of (top-left) EdU+ nuclei in zfp-1 (RNAi) and control colonies showed a significant reduction, contributing to the smaller colony size. Comparison of normalized EdU+ nuclei numbers (top-right) showed a non-significant difference, indicating that a similar proportion of neoblasts in the colony were cycling (Methods). Representative EdU labeling images (z-projection) are shown (Bottom). Statistical significance was assessed using Mann-Whitney two-tailed test (Methods). n.s., not significant, **** P < 0.0001. Scale = 100 µm.