The C. elegans gonadal sheath Sh1 cells associate selectively with a differentiating germ cell population in the proliferative zone

1 germ cell population in the proliferative zone 2 3 Xin Li, Noor Singh, Camille Miller, India Washington, Bintou Sosseh, Kacy Lynn Gordon 4 5 Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA 6 *Correspondence: kacy.gordon@unc.edu 7 Department of Biology, CB #3280, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, 8 USA 9 10 Abstract 11 The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath 12 cells that support germ cells as they mature from the stem-like mitotic state through meiosis, 13 gametogenesis and ovulation. Recently, we discovered that the distal-most Sh1 sheath cells 14 associate with mitotic germ cells as they exit the niche. Here we report that these distal sheath15 associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ 16 cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several 17 markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, 18 as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause 19 gonad morphology defects. However, such reagents with highly variable expression can be used to 20 determine the relative positions of the two Sh1 cells, one of which often extends further distal than the 21 other. 22 23 Introduction 24 The C. elegans hermaphrodite gonad is a fruitful system in which to study organogenesis, meiosis, 25 and stem cell niche biology. Recent work from our group (Gordon et al., 2020), used two 26 endogenously tagged alleles of genetically redundant innexin genes inx-8 and inx-9 to visualize the 27 somatic gonadal sheath of the C. elegans hermaphrodite. We discovered that the distal most pair of 28 sheath cells, called Sh1, lies immediately adjacent to the distal tip cell (DTC), which is the stem cell 29 niche of the germ line stem cells. Previously (based on electron microscopy and on cytoplasmic GFP 30 overexpression from transgenes active in the sheath (lim-7p::GFP) (Hall et al., 1999) or its progenitor 31 cells (lag-2p::GFP) (Killian and Hubbard, 2005)), Sh1 cells were thought to associate only with germ 32 cells well into the meiotic cell cycle, so our finding required a reimagining of the anatomy of the distal 33 gonad. 34 35 Here, we confirm that the Sh1 cells fall at the boundary of a population of germ cells in a stem-like 36 state, report other markers that label the Sh1 cells, and verify that these markers can be used to 37 assess gonad anatomy without unduly impacting the gonad itself. We also discuss reagents that are 38 not suitable markers of Sh1 cells, including an overexpressed, functional cell death receptor that is 39 used to mark Sh1 in a recent study (Tolkin et al., 2021). Finally, we consider best practices for using 40 endogenously tagged proteins for cell and developmental studies. 41 42 Results 43 Distal Sh1 associates with the population of germ cells that differentiate first when progression 44 through mitosis is halted or Notch signaling is lost 45 The DTC expresses the Notch ligand LAG-2, which is necessary to maintain the germ line stem cell 46 pool (Henderson et al., 1994). Recent work has shown that the active transcription of Notch targets 47 sygl-1 and lst-1 (Lee et al., 2019) and the accumulation of their proteins (Shin et al., 2017) is 48 restricted to the distal-most germ cells, describing a population of stem-like germ cells ~6-8 germ cell 49 diameters (~30-40 μm) from the distal end of the gonad. A similar spatial arrangement was found in 50 earlier work that used an emb-30 temperature-sensitive allele to arrest germ cell division, thus halting 51 the distal-to-proximal movement of germ cells and allowing cells to differentiate outside of the niche 52 and remain undifferentiated in the niche (Cinquin et al., 2010). Similar results were obtained using 53 glp-1 temperature sensitive alleles to stop Notch signal transduction and observe where and when 54 germ cells acquire markers of differentiation (Cinquin et al., 2010), though germ cell cycle also 55 influences the timing of differentiation (Fox and Schedl, 2015). Our recent work (Gordon et al., 2020) 56