Fig. 5. PLK-3 expression is restricted to the distal germline by piRNA function.

(A and B) Wild-type adult male germline expressing PLK-3::GFP (green) stained with DAPI (white). (A) PLK-3::GFP is expressed in the distal regions of the germline in the areas noted by the yellow arrowheads. (B) Zoomed in region of the distal germline from (A), displaying PLK-3::GFP expression between the progenitor zone and meiotic regions of the germline. (C and D) Wild-type adult male germline expressing PLK-3::GFP (green) and V5::mCherry::PRG-1 (magenta) stained with DAPI (white). (D) Zoomed in distal germline from (C), showing that PLK-3 expression is spatially expressed before and almost mutually exclusive with PRG-1 expression at the end of the progenitor zone, but PRG-1 expression is highest in the meiotic region. Inset 1 (yellow, dashed lines) shows a single TZ nucleus with visible PLK-3 perinuclear granules, whereas inset 2 (red, dashed lines) shows a pachytene nucleus with PRG-1 perinuclear granules. (E) Wild-type adult male germline stained with DAPI (white) and plk-3 HCR-FISH, showing expression of plk-3 mRNA throughout the distal regions with the mRNA expression significantly reduced in mid/late pachytene (red arrowhead). (F) A prg-1(null) adult male germline expressing PLK-3::GFP (green) stained with DAPI (white). Inset (yellow, dashed lines) displays single pachytene nucleus in the meiotic region expression PLK-3::GFP. (G) Schematic displaying the plk-3 coding sequence where 21ur-10935’s target sequence maps. Below, plk-3(Δpi) designates CRISPR-edited sequence with specific base changes noted (red, underlined). Predicted base-pairing within seed sequence (yellow box) and supplemental sequence (orange box) is noted. (H) Germline with PLK-3(Δpi)::GFP (green) stained with DAPI (white). Inset (yellow, dashed lines) displays pachytene nucleus in the meiotic region expressing PLK-3(Δpi)::GFP. Scale bars, 50 μm. Each experiment was conducted at least in triplicate and over at least 25 to 30 germlines analyzed each time.