Figure 3. Identification of genes regulated by Smed-pou4-2 using RNA-seq.
(A) Volcano plot of genes differentially expressed in pou4-2(RNAi) animals compared to controls (FC ≥1.4, p-adj ≤0.1). A subset of genes examined in this study is highlighted on the plot and demonstrates significant downregulation. (B) Co-localization analysis by double-fluorescence in situ hybridization reveals that 74.8% of pou4-2+ cells co-express pkd1L-2, and 28.4% co-express hmcn-1-L. White boxed cells shown in insets show high pou4-2 and terminal marker expression and are displayed at higher magnification. White arrowheads point to examples where terminal marker gene expression is much brighter than pou4-2 expression. White arrows mark pou4−2+ cells with high expression of pou4-2 and low expression of the terminal marker genes. Scale bar = 100 μm. (C) WISH of pkd1L-2 and hmcn-1-L in control and pou4-2(RNAi) 10 day regenerates. Terminal marker expression is strongly reduced in RNAi animals. Numbered red boxes demonstrate a population of scattered hmcn-1-L+ cells that persist following pou4-2 RNAi and are shown in corresponding zoomed-in insets. Blue arrows denote expression in the dorsal and peripheral ciliated stripes (dcs and pcs, respectively). Note that some pkd1L-2 and hmcn-1-L expression was detectable in regenerates (magenta arrows). Anterior is to the top. Scale bars = 200 μm; n ≥3 worms tested, with all samples displaying similar expression patterns.
Figure 3—figure supplement 1. Irradiation reveals that pou4−2+cells include progenitors.
