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

Figure 2. Sheath-expressed fluorescent proteins show consistency among endogenously tagged membrane proteins and greater variability in overexpressed transgenes.

(A) NK2571 qy78[mKate::inx-8]; cpIs122[lag-2p::mNeonGreen:: PLCδPH] N=21. (B) KLG019 qy79[GFP::inx9];naSi2 (channel not shown) N=16. (C) NK 2324 qy23[ina-1::mNG] N=26. (D) LP530 cp243[cam-1::mNG] N=21. (E) Box plots of Sh1 positions for all strains listed above and below, with fluorescent protein listed on the graph, including transgenes. (F) DG1575 tnIs6[lim-7p::GFP] N=20. (G) Strain DG5020 bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH] N=52 (note that mean and range agree with those reported in Tolkin et al., 2022). (H) KLG020 rlmIs5[lim-7p::GFP::CAAX];cpIs122 N=21. Purple gradient marks approximate extent of stem cell zone (Lee et al., 2019; Shin et al., 2017). See Figure 2—figure supplement 1 for images of minimum and maximum observed distances for all markers. Figure 2—figure supplement 2 shows comparisons across development of NK2571 and DG5020. All scale bars 10 µm.

Figure 2—source data 1. Source data used to generate plots of distal sheath measurements for strains shown in Figure 2.

Figure 2.

Figure 2—figure supplement 1. Endogenously tagged fluorescent proteins in the Sh1 membrane are less variable than overexpressed integrated transgenes.

Figure 2—figure supplement 1.

Minimum (left column) and maximum (right column) measurements of the distance between distal Sh1 and the distal end of the gonad for (A, A’) qy78[mKate::inx-8], (B, B’) qy79[GFP::inx9], (C, C’) qy23[ina-1::mNG], (D, D’) cp243[cam-1::mNG] (E, E’) tnIs6[lim-7::GFP], (F, F’) bcIs39[lim-7p::ced-1::GFP], (G, G’) rlmIs5[lim-7p::GFP::CAAX]. Arrowheads in G and G’ mark non-sheath cells positive for lim-7p::GFP::CAAX expression in the plane of the gonad and are unavoidably included in z-projections that capture the gonadal cell surface. Note especially in G how dim the Sh1 expression is at the distal extent, resembling what is sometimes seen for CED-1::GFP expression as reported by Figure 2—figure supplement 2B of Tolkin et al., 2022. In some cases, the selected images are near-minimum or near-maximum due to imaging artifacts like low illumination or sample movement in the true minimum or maximum images. All scale bars 10 µm.
Figure 2—figure supplement 2. Differences between qy78(mKate::inx-8) and bcIs39(lim-7p::ced-1::GFP) expression in the sheath appear at the L4-young adult transition.

Figure 2—figure supplement 2.

Side-by-side developmental comparisons between the two favored marker strains. Left column, DG5020 (bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH]); right column, NK2571 (qy78[mKate::inx-8]; cpIs122[lag-2p::mNeonGreen:: PLCδPH]). Number of worms examined for each strain at each stage given in the figure annotations. Note that both strains show a close association of the distal tip cell (DTC) and sheath cells during larval development; fluorescence signal from lim-7p::ced-1::GFP appears later in development than mKate::inx-8 signal (top row). During dorsal DTC migration, highly variable gaps often appear between the DTC and Sh1 in both marker strains (not pictured). Rapid Sh1 growth during this stage has been observed (Gordon et al., 2020), so we attribute these variable gaps to our taking snapshots of a dynamic process. By the end of gonad elongation, most Sh1 cells come to rest within a germ cell diameter of the DTC for both strains. The large gap between the DTC and Sh1 only appears in the lim-7p::ced-1::GFP strain after larval gonad migration is complete. This stage is also when the lim-7p::ced-1::GFP expressing sheath develops two other unique characteristics—holes over the germ cell bodies and a frilly distal edge with small, thin distal projections.