Figure 6. mex3-1 is required for epithelial turnover and regeneration as well as differentiation toward multiple tissues.

(A) RNAi animals were administered EdU 6 days after RNAi, and EdU⁺ labeling in the epithelium was quantified after a 7-day period. Single confocal planes are shown, with EdU⁺ cells in the epithelium indicated by arrows. Top panel scale bar, 100 μm; bottom panel scale bar, 50 μm. (B) RNAi animals amputated 7 days after RNAi were analyzed after 7 days regeneration by dFISH for stem cells and epithelial-associated genes. Single confocal planes or projections of head blastemas are shown, with anterior to the left. Dotted lines indicate animal boundary. Scale bar, 100 μm. (C) Diagram indicating regions of animal imaged and examined for quantification of lineage-restricted neoblast progeny. (D) Lineage-restricted progeny populations were quantified in intact worms 12 days after RNAi, using PIWI-1 immunolabeling and FISH for ovo (eyes), chat or coe (brain), FoxA (pharynx), and six1/2-2 (protonephridia). Single confocal planes are shown. Arrows indicate example double-positive cells. Magnified areas are indicated by dashed boxes and inset to the right of each image. Scale bar, 50 μm. (E) RNAi animals were administered BrdU 6 days after RNAi, and BrdU⁺ labeling in differentiated tissues (chat, brain; mat, gut; laminin, pharynx) was examined after a 5-day period. Single confocal planes are shown. Arrowheads indicate example double-positive cells. Scale bar, 50 μm. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test). (F) Quantification of stem cells (piwi-1⁺PIWI-1⁺) and immediate postmitotic stem cell descendants (piwi-1⁻PIWI-1⁺) were performed 6 days after RNAi, in head, pre-pharyngeal, and tail regions. Shown are single confocal planes from the tail region. Magnified areas are indicated by dashed boxes. The proportion of postmitotic descendants to stem cells between mex3-1(RNAi) and control worms differed significantly (p < 0.001, non-linear regression analysis). Scale bar, 50 μm on left panels, 10 μm on right panels. All counts were performed in 5–10 animals.

DOI: http://dx.doi.org/10.7554/eLife.07025.017

Figure 6—figure supplement 1. mex3-1 RNAi impairs brain differentiation.

(A) Expression of mex3-1 in neural-restricted neoblast progeny by mex3-1 and chat dFISH with PIWI-1 immunolabeling. Percentage of chat⁺PIWI-1⁺ cells which express mex3-1 is indicated at top-right of panel. Arrows indicate example triple-labeled cells. Dashed box indicates area of magnified panels. Left scale bar, 50 μm; right scale bar, 10 μm. (B) Animals were administered BrdU at 6 days after RNAi and assessed at 5 days post-BrdU. Scale bar, 100 μm. All images shown are single confocal planes.

DOI: http://dx.doi.org/10.7554/eLife.07025.018

Figure 6—figure supplement 2. Differentiation in p53(RNAi) animals.

(A) Stem cell (piwi-1) and epithelial progenitor (prog-1) populations were examined by WISH in RNAi animals. Scale bar, 200 μm. (B) Eye and neural lineage-restricted neoblast progeny were quantified in RNAi animals by FISH (ovo, eye; chat, brain) with PIWI-1 immunolabeling. Arrows indicate example double-positive cells. Scale bars, 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.07025.019