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 Caenorhabditis elegans. We show that a reduction of soma–germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions—lacking somatic gonadal cell coverage of germ cells—are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor 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 E ). These markers colocalize in a substantial fraction of animals, as has been reported recently ( Tolkin et al, 2022 , see Figure 2—figure supplements 1 and 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.…”

“…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.…”

“…( 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.…”

“…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, 2022 ). 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 ).…”

“…Here, we confirm that the Sh1 cells fall at the boundary of a population of germ cells in a stem-like state, report other markers that label the Sh1 cells, and verify that these markers can be used to assess gonad anatomy without unduly impacting the gonad itself. We also discuss reagents that are not suitable markers of Sh1 cells, including an overexpressed, functional cell death receptor that is used to mark Sh1 in a recent study ( Tolkin et al, 2022 ). Finally, we consider best practices for using endogenously tagged proteins for cell and developmental studies.…”