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. 2022 Sep 12;11:e75497. doi: 10.7554/eLife.75497

Figure 3. lim-7p::CED-1::GFP has variable expression intensity that conceals distal position of Sh1.

(A) Plot of distal position vs. fluorescence intensity in arbitrary units of CED-1::GFP at the distal limit of its domain in N=30 DG5020 bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH] animals. Dashed black line: all of the lowly expressing gonads (under ~400 AU, or <30% maximum brightness of brightest sample) have a distal tip cell (DTC)-Sh1 interface detected. (B) DG5020 sample in which disparate expression levels in the two Sh1 cells of a single gonad arm obscure detection of the DTC-Sh1 interface. The GFP channel is scaled automatically in B; B’ is scaled to saturate the brightest pixels and reveal the dim second Sh1 cell. Dashed yellow link marks the edge of the bright Sh1 cell. (C) Schematic showing Sh1 pair configuration over distal germline, with the distal extent of Sh1p uncertain in superficial projection. The two Sh1 cells of a pair descend from the anterior and posterior daughters of Z1 and Z4, so the two Sh1 cells are here labeled Sh1a and Sh1p (arbitrarily). Top, superficial view. Bottom, side view. (D) DG5131 qy78[mKate::inx-8]; bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH] sample in which one Sh1 cell contacts the DTC around the circumference of the germline and the other Sh1 cell lies at some distance from the distal end. Gray boxes and numbers mark planes and landmarks shown in (E). (E) Five cross sections through gonad in (E) made by projecting through two 1 µm re-slices at the positions shown by gray boxes in (D). Same analysis for DG5020 shown in Figure 3—figure supplement 1. (F) Same worm as in (D,E); signal from endogenously tagged allele qy78[mKate::inx-8] more uniformly labels the Sh1 cells, obscuring their individual shapes. All scale bars 10 µm.

Figure 3—source data 1. Source data used to generate plots of distal sheath position and fluorescence intensity measurements for samples shown in Figure 3A and Figure 4D.

Figure 3.

Figure 3—figure supplement 1. The Sh1 cells of a pair can take two distinct configurations over the distal germline.

Figure 3—figure supplement 1.

(A) Example of a gonad from DG5020 lim-7p::ced-1::GFP animal with dramatically different CED-1::GFP signals revealing the shapes of the two Sh1 cells of the pair. Gray boxes show planes depicted in (B). (B) Three cross sections through gonad in (A) made by maximum projection through two 1 µm re-slices (FIJI) at the positions shown by gray boxes in (A). Dashed yellow and white lines mark the two Sh1 cells. Depending on the proximodistal position of the gonad, one or the other Sh1 cell may surround more of the germline. (C) Example of another gonad from DG5020. (D) Three cross sections through gonad in (C) made by projecting through two 1 µm re-slices at the positions shown by gray boxes in (C). (E,F) Gonads from strain DG5131 with merged images on top, CED-1::GFP channel in the middle, and mKate::INX-8 and distal tip marker channel on the bottom. Yellow outlines show regions of interest in which fluorescence intensity was measured. bg = background, subtracted from fluorescence intensity measured in each of the two Sh1 cells, which express CED-1::GFP at disparate levels. (E’ and F’) Insets from E and F. In both cases, mKate::INX-8 signal is half as strong in the Sh1 cells with more CED-1::GFP. Note also in E and F that mKate::INX-8 and bright CED-1::GFP mark a different distal extent of Sh1. All scale bars 10 µm.