Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool

Abstract

Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in the nematode Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional signaling causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We show that a somatically expressed innexin fusion protein, which was used as marker in a prior study asserting that the wild type lacked a bare region between the distal tip cell (DTC) and Sh1, encodes a poisonous gap junction subunit. We determine that, contrary to the model put forth in the prior study based on this marker, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool or of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone

“…The Sh1 positions become even more clear when lim-7p::ced-1::GFP is coexpressed with the mKate-tagged innexin inx-8(qy78) in strain DG5131 (Figure 3D and 3E). These markers colocalize in a substantial fraction of animals, as has been reported recently ((Tolkin et al, 2021), see Figure 2 Supplement 2 therein). In the animals that have a discrepancy between GFP and mKate localization in Sh1, the difference in expression reveals an unexpected cell boundary between the two Sh1 cells.…”

“…The discrepancy in apparent Sh1 position when two Sh1 cells express different amounts of CED-1::GFP and when CED-1::GFP is coexpressed with mKate::INX-8 provides definitive evidence that CED-1::GFP sometimes fails to label the entire distal sheath (the same phenomenon is reported in Figure 2 Figure Supplement 2B in the recent study (Tolkin et al, 2021)). Furthermore, the defects caused in gonads overexpressing this functional cell death receptor suggests its localization to the distal Sh1 membrane at high levels is not well-tolerated.…”

“…(A) NK2571 qy78[mKate::inx-8]; cpIs122[lag-2p::mNeonGreen:: PLC δPH ] 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, 2021)) (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).…”

“…We find this marker to be unreliable in the distal region of the cell, and to cause gonad defects especially but not only when combined with endogenously tagged inx-8(qy78) . A recent study (Tolkin et al, 2021) uses this transgene in most of the backgrounds analyzed (sometimes detecting the CED-1::GFP by anti-GFP antibody staining, which appears to amplify the variability of the marker), so we find this problematic reagent to undermine that study’s conclusions.…”

“…The first is a lim-7 promoter-driven cytoplasmic GFP that was used to label the Sh1 cell in a foundational study of the C. elegans hermaphrodite gonad, tnIs6[lim-7::GFP] (Hall et al, 1999) (Figure 2F). The second is a lim-7 promoter-driven functional cell death receptor tagged with GFP, bcIs39[lim-7p::ced-1::GFP] (Zhou et al, 2001), which is the basis of a recent study that reports a more proximal boundary of Sh1 (Figure 2G, strain DG5020 (Tolkin et al, 2021)). The third is a lim-7 promoter-driven membrane-localized GFP made by us to mark the sheath cell membrane without tagging an endogenous protein, rlmIs5[lim-7p::GFP::CAAX] (Figure 2H).…”