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

  1. Xin Li
  2. Noor Singh
  3. Camille Miller
  4. India Washington
  5. Bintou Sosseh
  6. Kacy Lynn Gordon  Is a corresponding author
  1. Department of Biology, The University of North Carolina at Chapel Hill, United States

Abstract

The Caenorhabditis elegans adult hermaphrodite germline is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis, and ovulation. Recently, we discovered that the distal Sh1 sheath cells associate with mitotic germ cells as they exit the niche Gordon et al., 2020. Here, we report that these sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other.

Editor's evaluation

This work extends the previous findings by the authors that suggested the lack of 'bare region' in the C. elegans gonad, which was previously postulated to exist between germ cells that are encapsulated by the distal tip cell and those that are encapsulated by sheath cells. The authors addressed the concerns posed by Tolkin et al. that proposed that the bare region does exist. However, discrepancies remain between the current manuscript and the manuscript by Tolkin et al., which should be resolved in the field in the future. Overall, the work presented here is important and of broad interest as it concerns the regulation of the stem cell niche and how cells that are destined to differentiate exit the niche and proceed to differentiation by interacting with the stromal cells.

https://doi.org/10.7554/eLife.75497.sa0

Introduction

The Caenorhabditis elegans hermaphrodite gonad is a fruitful system in which to study organogenesis, meiosis, and stem cell niche biology. Recent work from our group (Gordon et al., 2020) used two endogenously tagged alleles of genetically redundant innexin genes inx-8 and inx-9 to visualize the somatic gonadal sheath of the C. elegans hermaphrodite. We discovered that the distal most pair of sheath cells, called Sh1, lies immediately adjacent to the distal tip cell (DTC), which is the stem cell niche of the germline stem cells. Previously (based on electron microscopy and on cytoplasmic GFP overexpression from transgenes active in the sheath [lim-7p::GFP] [Hall et al., 1999] or its progenitor cells [lag-2p::GFP] [Killian and Hubbard, 2005]), Sh1 cells were thought to associate only with germ cells well into the meiotic cell cycle, so our finding required a reimagining of the anatomy of the distal gonad.

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.

Results

Distal Sh1 associates with the population of germ cells that differentiate first when progression through mitosis is halted or Notch signaling is lost

Our first experiment addresses in a new way the question of what type of germ cells associate with the distal Sh1 cell. The DTC expresses the Notch ligand LAG-2, which is necessary to maintain the germline stem cell pool (Henderson et al., 1994). Recent work has shown that the active transcription of Notch targets sygl-1 and lst-1 (Lee et al., 2019) and the accumulation of their proteins (Shin et al., 2017) is restricted to the distal-most germ cells, describing a population of stem-like germ cells ~6–8 germ cell diameters (~25–35 µm) from the distal end of the gonad. Our recent work (Gordon et al., 2020) reported that the position of Sh1 coincides with sygl-1 promoter’s activation boundary on one side and the accumulation of the meiotic entry protein GLD-1 on the other, consistent with the hypothesis that the distal edge of Sh1 falls at the boundary of that stem-like cell population, ~30 µm from the distal end of the gonad.

A similarly positioned stem-like germ cell population was found in earlier work that used temperature-sensitive alleles to perturb germ cell fate or progress through the cell cycle (Cinquin et al., 2010). The readout was germ cell fate as determined in one of two ways. Anti-phosphohistone H3 staining of proliferative cells and GLD-1 antibody costaining for cells accumulating meiotic factors shows where cells are dividing and beginning to differentiate, respectively. Alternatively, the ‘transition zone’ in germ cell nuclear morphology between ‘mitotic’ and ‘meiotic’ zones can be visualized by the presence of crescent-shaped nuclei of meiotic prophase observed by DAPI staining. We used this latter method of visualizing nuclear morphology.

We repeated these experiments in strains that have tagged innexins to mark the distal sheath Sh1 cells to ask which population(s) of germ cells are associated with Sh1. Here, we describe the original findings and their interpretations, and then our new findings. First, an emb-30 temperature-sensitive allele is known to cause germ cell division to arrest at the metaphase-anaphase transition, thus halting the distal-to-proximal movement of germ cells that is driven by the proliferation of more distal cells (Cinquin et al., 2010). In a wild-type gonad, germ cells differentiate (enter and progress through the meiotic cell cycle) as they move from distal to proximal (Figure 1A, left). In emb-30(ts) gonads, a shift to the restrictive temperature causes proliferation to halt and germ cells to remain stationary within the gonad (Figure 1A, right). These cells can now differentiate in place—or remain in the undifferentiated state—depending on their exposure to the stemness cue. Germ cells that remain in the niche at the distal end of the gonad do not differentiate after 15 hr at the restrictive temperature, while more proximal germ cells do differentiate. Nuclear morphology differs between these two regions of the germline.

Figure 1 with 1 supplement see all
The Sh1 cells associate with proliferative germ cells that are on the path to differentiation.

(A) Schematic of hypothesis for emb-30(tn377) experiment. Germ cell (gc) nuclei shown in magenta, somatic gonad cells shown in green (distal tip cell [DTC]), and transparent green (Sh1). (B) Gonads from KLG023 emb-30(tn377);qy78;cpIs122 worms reared at permissive (left column) and restrictive (right column) temperatures. Top, merged image. Middle, mKate::INX-8 labeling Sh1 (edge outlined with yellow dashed line). Bottom, DAPI staining labeling all nuclei with pink arrow marking gc transition and same yellow dashed line as in middle image showing Sh1 edge. (C) Schematic of hypothesis for glp-1(ts) experiment. (D) Gonads from KLG022 glp-1(bn18);qy79;naSi2 worms reared at permissive (left column) and restrictive (right column) temperatures. Top, merged image. Middle, GFP::INX-9 labeling DTC (outlined in white) and Sh1 (edge outlined with yellow dashed line). Bottom, germ cell histone mCherry (naSi2[mex-5p::H2B::mCherry]) with pink arrow showing gc transition and same yellow dashed line as in middle image showing Sh1 edge. Note that the glp-1(bn18) allele is not fully wild type at permissive temperatures and is known to have a shortened proliferative zone (Fox and Schedl, 2015). (E) Box plots overlaid with all datapoints measuring the distal position of Sh1 and the position of the transition zone in germ cell nuclear morphology. Permissive temperature shown in blue; restrictive temperature shown in red. Permissive emb-30 N=30; restrictive emb-30 N=34. Permissive glp-1 N=18; restrictive glp-1 N=21. A one-way ANOVA to assess the effect of temperature on proximodistal position of gonad features was performed, and was significant for emb-30: F3,124=134.5, p<0.0001. Tukey’s multiple comparison test found that the mean values of the positions of Sh1 and the germ cell transition zone were significantly different at the permissive temperature (mean difference of –45.25 μm, 95% CI –52.38 to –38.12 μm, p<0.0001), but not at the restrictive temperature (mean difference of –2.30 μm, 95% CI –9.00 to 4.40 μm, p=0.808). The position of the germ cell transition zone differed at the permissive vs. restrictive temperatures (mean difference of 42.87 μm, 95% CI 35.95 to 49.79 μm, p<0.0001), but the Sh1 position did not (mean difference of –0.078 μm, 95% CI –7.00 to 6.84 μm, p>0.9999). This pattern is observed across replicates and various controls (Figure 1—figure supplement 1). Similar results were obtained for glp-1: F3,74=52.84, p<0.0001. Tukey’s multiple comparison test found that the mean values of the positions of Sh1 and the germ cell transition zone were significantly different at the permissive temperature (mean difference of –35.51 µm, 95% CI –44.59 to –26.43 µm, p<0.0001) but not at the restrictive temperature (mean difference of 2.514 µm, 95% CI –5.892 to 10.92 µm, p=0.861). The position of the germ cell transition zone differed at permissive vs. restrictive temperatures (mean difference of 36.02 µm, 95% CI 27.27 to 44.77 µm, p<0.0001), but the Sh1 position did not (mean difference of –1.997 µm, 95% CI –10.75 to 6.753 µm, p=0.9318). All scale bars 10 µm.

Figure 1—source data 1

Source data used to generate plots of distal sheath and germ cell transition zone measurements at permissive and restrictive temperatures for mutant strains shown in Figure 1.

https://cdn.elifesciences.org/articles/75497/elife-75497-fig1-data1-v1.xlsx

We hypothesized that the transition in nuclear morphology in emb-30(tn377) animals would shift proximally after 15 hr at the restrictive temperature (as had previously been observed), to ultimately fall at the distal position of the Sh1 cell as visualized by mKate::INX-8; we hypothesize that the position of the Sh1 cell would itself not be affected by the temperature shift. Indeed, this is what we found (Figure 1B and E), supporting our prior conclusion that there is germ cell fate asymmetry across the DTC-Sh1 boundary. These results are independent of culture time of controls at the permissive temperature (Figure 1—figure supplement 1). The Sh1 cells cover proliferative germ cells outside the niche that are closer to differentiating than those under the DTC.

The second set of experiments using temperature-sensitive alleles to reveal differences in germ cell fate along the distal-proximal axis uses glp-1(ts) alleles to stop Notch signal transduction and observe where and when germ cells acquire features of differentiation (Figure 1C). The same study (Cinquin et al., 2010) found that the glp-1(q224) temperature-sensitive allele reared at the restrictive temperature over a 9 hr time course showed a progressively shrinking mitotic zone (as assessed by nuclear morphology of DAPI stained gonads) until hour ~5.5, at which time the remaining distal most ~5 rows of germ cells differentiate as a pool. A subsequent study (Fox and Schedl, 2015) used a similar approach (but with the glp-1(bn18) temperature-sensitive allele, slightly different timing, and antibody staining to determine cell fate) and found a similar result, and additionally discovered that progress through the cell cycle influenced the precise timing of germ cell differentiation.

We used the glp-1(bn18) allele to deactivate Notch signaling in a strain with GFP::INX-9 to visualize the Sh1 cells and the fluorescent histone marker naSi2(mex-5p::H2B::mCherry) to visualize germ cell nuclei (Figure 1D and E). We hypothesized that after 6 hr at the restrictive temperature, only the distal-most pool of stem-like cells will not have taken on the crescent-shaped nuclear morphology of meiotic germ cells, while the germ cells under Sh1 will have entered the meiotic cell cycle. We predicted that the Sh1 cell would not change its position across this time interval. Indeed, this is what we found, further supporting our hypothesis that the Sh1-associated germ cells are closer to differentiation than those under the DTC, which are the last to differentiate.

Results from these temperature-sensitive mutants confirm what the markers of germ cell fate revealed in Gordon et al., 2020, which is that the Sh1 cell associates with germ cells in the proliferative zone that have left the stem cell niche and are on the path to differentiation, while the stem-like germ cells lie immediately distal to the Sh1 cell at its interface with the DTC.

Different endogenously tagged membrane proteins reveal a distal position of Sh1

These experiments made use of the endogenously N-terminal tagged inx-8(qy78[mKate::inx-8]) and inx-9(qy79[GFP::inx-9]) alleles (Figure 2A and B) generated by Gordon et al., 2020. Both tagged proteins are highly specific for the somatic gonad throughout development; in the adult, their expression differentiates, with INX-8 signal diminishing from the DTC and INX-9 signal persisting (see white DTC outline in Figure 1D). We have since identified additional endogenous fluorescent-protein-tagged alleles that show expression in the gonadal sheath cells and localize in or near the cell membrane. One of these, ina-1(qy23[ina-1::mNeonGreen]) (Figure 2C), was briefly reported in Gordon et al., 2020. We found another that marks the sheath, cam-1(cp243[cam-1::mNeonGreen]) (Heppert et al., 2018; Figure 2D). For both tagged innexins as well as ina-1::mNG and cam-1::mNG, we find that the Sh1 cell has a distal boundary that either displays a measurable interface with the DTC or is so located as to be consistent with such a boundary (where the DTC is not marked by the endogenous protein). The position of this boundary (<40 µm, or ~8 germ cell diameters) coincides with the domain in which germ cells leave the stem cell niche (Lee et al., 2019) (purple shading in Figure 2E). We have not yet found a counterexample of an endogenously tagged, membrane-associated protein in Sh1 that demarcates an apparent Sh1 cell boundary at a great distance from the distal end of the gonad in young adults.

Figure 2 with 2 supplements see all
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.

https://cdn.elifesciences.org/articles/75497/elife-75497-fig2-data1-v1.csv

Overexpressed transgenic markers vary in distal position and expression levels

Three integrated array transgene markers that drive overexpression of fluorescent proteins in the sheath were also analyzed. 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). The ranges of the distal edge of GFP localization for all three strains overlap with what we observed for the four endogenously tagged proteins, but are far more variable, as overexpressed transgenes are known to be (Evans, 2006; Figure 2E–H, and Figure 2—figure supplement 1). Patterns are more similar at earlier developmental stages (Figure 2—figure supplement 2).

To untangle this variance, we examined individual worms for evidence of a DTC-Sh1 interface. About half of the scoreable lim-7p::ced-1::GFP gonads (strain DG5020) show a DTC-Sh1 interface, and half show a bare region (Figure 3A). We further broke down this dataset by fluorescence intensity of distal CED-1::GFP signal. Strikingly, among animals under a threshold of expression intensity of ~400 AU (less than 1/3 as bright as the brightest GFP samples), the incidence of a DTC-Sh1 interface was 100% (10/10, as opposed to 15/30 for the whole dataset, Figure 3A). On the other extreme, gonads with stronger CED-1::GFP signal were more likely to have a farther proximal boundary of CED-1::GFP localization. In samples for which CED-1::GFP signal terminates at a great distance from the distal end of the gonad, there are two possible explanations. Either in those animals, the Sh1 position is farther proximal than in animals with other markers, or else CED-1::GFP fails to localize to the edge of the Sh1 cell pair.

Figure 3 with 1 supplement see all
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.

https://cdn.elifesciences.org/articles/75497/elife-75497-fig3-data1-v1.xlsx

Expression differences between Sh1 cells in a pair can conceal distal extent of the sheath

We observed a pattern in a subset of gonads where the two Sh1 cells of a pair had dramatically different levels of CED-1::GFP signal, and these cells had different terminal positions on the distal-proximal axis (Figure 3B and B’). Exposure time and excitation laser power during image acquisition and subsequent scaling of the resulting image determine whether or not the signal in the lowly expressing cell is readily apparent (Figure 3B vs B’). In some cases, the brightness of the other Sh1 cell and the nearby proximal gonad makes the dimmer Sh1 cell nearly impossible to detect. Variable expression levels and even complete silencing of C. elegans transgenes are well-known phenomena (Evans, 2006). It was not known, however, that the two Sh1 cells of a pair could assume such different configurations over the distal germline (Figure 3C, and see Figure 2H for the same pattern in the lim-7p::GFP::CAAX transgenic strain).

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. We imaged 19 gonads from the coexpressing strain DG5131 through their full thickness. Of those, 4/19 had severe gonad morphology defects (see next section). Of the 15 morphologically normal gonads, 6/15 had discrepancies in CED-1::GFP and mKate::INX-8 signal. In 3/6 such cases, one Sh1 cell makes up the entire DTC-Sh1 interface, with the other terminating at a greater distance from the distal end. In the other 3/6 of cases, both Sh1 cells border the DTC. Fluorescence signal from mKate::INX-8 alone does not allow these cell borders to be detected because that marker is more consistently expressed across the Sh1 cells (Figure 3F).

The variability of the lim-7p::ced-1::GFP transgene allowed us to perform something like a mosaic analysis when the two Sh1 cells have very different expression levels but the dimmer cell is still visible (N=31/53 morphologically normal DG5020 gonads imaged to full depth, Figure 3—figure supplement 1A-D). Where the borders of the two Sh1 cells can be distinguished, one cell extends at least 20 µm farther distal than the other in 23/31 cases; five additional gonads have expression in only one Sh1 cell that terminates at a great distance (>70 µm) from the distal end. The edges of dimly expressing Sh1 cells can be difficult to resolve. A similar phenomenon was observed when the cytoplasmic GFP of tnIs6[lim-7p::GFP] was coexpressed with qy78[mKate::inx-8] (Gordon et al., 2020; Figure 1—figure supplement 1 therein). Of note, the N-terminal mKate::INX-8 and GFP::INX-9 tags are most likely extracellular based on the innexin-6 channel structure determined by cryo-EM (Oshima et al., 2016), so there is reason to suspect their localization at the cell membrane will be regulated differently than that of intracellular GFPs.

Additionally, we noticed that in DG5131 gonads where the two Sh1 cells have very different CED-1::GFP expression levels, sometimes mKate::INX-8 is missing from the membrane in Sh1 cells with strong CED-1::GFP signal (Figure 3—figure supplement 1E and F). Subtracting background, we find that there is a 50% reduction in tagged INX-8 in such membrane regions. Since mKate::INX-8 is a genomically encoded, functional protein, such disruption likely impacts endogenous protein function. This observation hints at a synthetic interaction between the two fluorescent sheath membrane proteins.

Overexpression of CED-1::GFP transgene is correlated with gonad abnormalities

We therefore asked whether there was further evidence of a synthetic interaction between lim-7p::ced-1::GFP and inx-8(qy78). First, we found evidence that suggests that lim-7p::ced-1::GFP is damaging to the animals with or without qy78. In the strain that expresses lim-7p::ced-1::GFP and not qy78 (strain DG5020), roughly 20% of the animals had profound gonad migration defects in one gonad arm (Figure 4A and C). We also observe such defects in the DG5131 strain that combines qy78[mKate::inx-8] with the lim-7p::ced-1::GFP transgene (Figure 4B, 4/19 or 21% of animals), so we cannot attribute this defect to a spontaneous mutation arising in a single population in transit. We have not observed such morphological defects in the original strain bearing qy78, nor in any other strain we have studied. The lim-7p::ced-1::GFP transgene seems to cause incompletely penetrant gonad morphology defects.

lim-7p::ced-1::GFP is correlated with gonad defects.

(A) Example of gonad morphology defect in DG5020 bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH] strain, in which the gonad failed to turn. Gut outlined in dashed shape; magenta puncta in that domain are autofluorescent gut granules. (B) Example of gonad morphology defect in DG5131 qy78[mKate::inx-8]; bcIs39[lim-7p::CED-1::GFP]; naIs37[lag-2p::mCherry-PH] strain, in which gonad turned once and arrested without elongating along the dorsal body wall. Schematics in A and B show wild-type gonad morphology with two turns and a distal tip cell (DTC) that arrives at the dorsal midbody, left, beside schematics of defective gonad migration shown in micrographs. (C) Relative proportions of phenotypes observed in DG5020 animals (N=72). (D) Boxplot comparing fluorescence intensity for coexpressing strain DG5131 in addition to data shown in Figure 3 for DG5020. Fluorescence intensity of the lim-7p::ced-1::GFP transgene in this background is statistically indistinguishable from expression levels of this transgene in an otherwise wild-type background in the subset of samples that display a DTC-Sh1 interface, shown here segregated from samples from this strain that show a gap between the DTC and Sh1 cells. DG5131 N=17. DG5020 gap N=13. DG5020 interface N=17. A one-way ANOVA to determine the effect of category (genotype or presence of an interface) and fluorescence intensity was performed and was significant, F2,44=7.70, p=0.001. Tukey’s multiple comparison test finds that the mean fluorescence intensity of DG5020 gonads with a DTC-Sh1 interface differs from DG5020 gonads with a gap between Sh1 and the distal end (p=0.002) and does not differ from DG5131 worms (p=0.908). (E) Gonad from DG5131 strain with white arrowheads indicating aberrant engulfment of germ cells in the distal gonad. Closer to the distal end, a large mass of germ cells showing substantial localization of the CED-1::GFP protein may also reflect ectopic engulfment. Schematics show location of germ cell engulfment in wild-type gonads on the left and locations of the features marked in the micrograph in E on the right. Scale bars in A and B, 10 µm; scale bar in E, 25 µm.

Figure 4—source data 1

Classifications of 72 gonads from strain DG5020 that display a defect, a gap, or an interface used to generate pie chart in Figure 4C.

https://cdn.elifesciences.org/articles/75497/elife-75497-fig4-data1-v1.xlsx

Whether or not overexpressed CED-1::GFP also disrupts the localization of untagged innexin proteins or other endogenous sheath membrane proteins as it does the tagged mKate::INX-8, and whether such disruption explains the gonad migration defects we observe for this allele, we currently cannot say. In many of these qy78; lim-7p::ced-1::GFP coexpressing animals (strain DG5131), the intensity of CED-1::GFP is notably low (Figure 4D). Lower expression levels of the CED-1::GFP fusion protein, with or without qy78 in the background, appear more likely to reveal the distal Sh1 cell (Figure 3A and Figure 4D). This could either be because the absence of competing bright signal makes it easier to detect dimly expressing distal Sh1, or because high levels of the transgene product are not tolerated in the distal Sh1 cell. The overexpression of the functional cell death receptor CED-1, and not just the overexpressed membrane-localized GFP, could contribute to the defects observed in this strain. We sometimes observe abnormal sheath membrane protrusions that may result from aberrant engulfment of distal germ cells by the sheath (Figure 4E).

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 3B in the recent study Tolkin et al., 2022). Furthermore, the defects caused in gonads overexpressing this functional cell death receptor suggests that its localization to the distal Sh1 membrane at high levels is not well tolerated. We therefore conclude that lim-7p::ced-1::GFP is an unacceptable marker of distal Sh1.

Assessing sheath markers for evidence of gonad disruption—brood size

Just because lim-7p::ced-1::GFP is a poor marker of the distal sheath does not, however, relieve concerns that the endogenously tagged innexins mKate::INX-8 and GFP::INX-9 are altering the gonad. A control for tagged innexin function was originally carried out (Gordon et al., 2020). Briefly, a careful genetic analysis (Starich et al., 2014) reported that the single mutant inx-9(ok1502) is fertile, but the inx-8(tn1474); inx-9(ok1502) double mutant is sterile. Therefore, attempts to use CRISPR/Cas9 to introduce a fluorescent tag in the inx-8 locus were first performed in the inx-9(ok1502) background, and only once a fertile edited strain was recovered was the same edit introduced into the otherwise wild-type genetic background. We conducted brood size assays for strains discussed in this study, including the DG5131 strain containing both lim-7p::ced-1::GFP and the tagged innexin qy78[mKate::inx-8] that was imaged and analyzed by Tolkin et al., 2022, but not assayed for brood size (Table 1).

Table 1
Brood size assays.
Strain nameFull genotypeLive brood*Reduction vs. wt %Unhatched eggsEmbryonic lethality %
N2Wild type295±39 (n=57)NANANA
KLG019qy79[GFP::inx-9];nasi2, §226±22 (n=13)23%165±5841 ± 8%
NK2571qy78[mKate::inx-8];cpIs122§220±41 (n=15)25%20±149 ± 5%
DG5020bcIs39[lim-7p::ced-1::GFP];naIs37§202±29 (n=12)32%62±4720 ± 14%
DG5131qy78[mKate::inx-8];bcIs39[lim-7p::ced-1::GFP];naIs37§187±45 (n=14)37%40±2518 ± 11%
LP530cp243[cam-1::mNG]260±31 (n=10)12%NANA
NK2324qy23[ina-1::mNG]237±37 (n=8)20%NANA
  1. *

    Viable offspring that hatch from a single parent.

  2. N2 numbers come from multiple trials, not all of which were scored for embryonic lethality, including the trial in which ina-1(qy23) and cam-1(cp243) were counted.

  3. qy79[GFP::inx-9] allele in strains NK2572 and NK2573 from Gordon et al., 2020, with germ cell nuclear marker naSi2; this combination of alleles was used in the cross to glp-1(bn18) in Figure 1D.

  4. §

    Full transgene descriptions in Methods for germ cell (naSi2) and DTC (cpIs122, naIs37) markers.

  5. See Appendix 1—table 1 for replicates and statistical analysis of NK2571 and DG5020.

We find reductions in brood size for all of the strains under investigation, including a reduced brood size and notable embryonic lethality in two strains (DG5020 and DG5131) carrying the lim-7p::ced-1::GFP transgene. Interestingly, despite being genetically redundant genes (Starich et al., 2014) tagged in highly similar ways, and having similar live brood sizes, our endogenously tagged inx-8(qy78) and inx-9(qy79) strains had dramatically different degrees of embryonic lethality, with qy79 producing over 150 unhatched eggs per worm. All of the fluorescently marked strains have mildly to moderately reduced brood sizes. On the basis of brood size alone, there is not a strong reason to prefer one of these markers over another.

Assessing sheath markers for evidence of gonad disruption—proliferative zone

Because brood size is an emergent property of many gonad, germline, embryonic, and systemic processes (including gonadogenesis, stem cell maintenance, regulation of meiosis, spermatogenesis, oogenesis, metabolism, ovulation, and embryogenesis), defects in brood size are not a direct proxy for dysregulation of the germline proliferative zone. We therefore turned our attention back to the distal gonad and asked whether the strains with fluorescent sheath markers have abnormalities in several metrics (Figure 5A). The length of the proliferative zone differs among strains (as measured by DAPI staining of germ cell nuclei to detect and measure the length of the germline distal to crescent-shaped nuclei of meiosis I, Hubbard, 2007; Figure 5A, C and D). The NK2571 strain with the tagged innexin inx-8(qy78) and DTC marker has a normally patterned distal germline (average proliferative zone length of 106 µm, or ~26 germ cell diameters) that is indistinguishable from wild-type N2 (average of 109 µm or ~27 germ cell diameters, Figure 5C, C’ , and D). Excluding worms with gross morphology defects, the DG5020 strain bearing a DTC marker and lim-7p::ced-1::GFP has a measurably shorter distal germline (average of 91 µm, or ~23 germ cell diameters, Figure 5C” , and D). In the DG5131 strain that combines these alleles, the distal germline is notably shortened (average of 79 µm or ~20 germ cell diameters, Figure 5C”’ , and D). This is comparable to the defect caused by the glp-1(bn18) allele at the permissive temperature shown in Figure 1E. Abnormal distal gonad patterning provides further evidence that a synthetic interaction between the lim-7p::ced-1::GFP transgene and the qy78 allele—not the qy78 allele alone—is responsible for the shorter proliferative zone observed for strain DG5131 (in agreement with Figure 4 from Tolkin et al., 2022).

A synthetic interaction between lim-7p::ced-1::GFP and the tagged innexin qy78 shortens the proliferative zone.

(A) Illustration of measurements made for Figure 5. (B) Number of mitotic figures observed in DAPI stained animals of the four strains. Numbers of dividing cells and gonads examined are as follows: wild-type N2 (N=311 dividing cells/67 gonads), the NK2571 strain with the tagged innexin qy78 (N=184 dividing cells/36 gonads), the DG5020 strain with lim-7p::ced-1::gfp (N=105 dividing cells/31 gonads), the DG5131 strain combining these sheath markers (N=136 dividing cells/42 gonads). A one-way ANOVA to determine the effect of genotype on number of mitotic figures was significant F3, 172=7.081, p=0.0002. Tukey’s multiple comparison test revealed that NK2571 did not differ from wild type (mean difference –0.47 cells per gonad, 95% CI –1.65 to 0.71, p=0.7291), DG5020 differed from wild type by 1.26 germ cells per gonad, 95% CI 0.02–2.50, p=0.0453, and DG5131 differed from wild type (mean difference of 1.40 cells per gonad, 95% 0.28–2.52, p=0.0075). (C-C””) DAPI stained distal gonads for measurement of proliferative zone for the four strains. Asterisk marks tip of gonad, dashed line marks example of lengths measured. (C) Wild-type N2 (N=68), (C’) the NK2571 strain with the tagged innexin qy78 (N=49), (C”) the DG5020 strain with lim-7p::ced-1::gfp (N=40), (C”’) and the DG5131 strain combining these sheath markers (N=45). Asterisk marks tip of gonad. (D) Plots of proliferative zone length (left) and whole gonad length (right) for the four strains. A one-way ANOVA to determine the effect of genotype on length of proliferative zone was significant F3,198=49.15, p<0.0001. Tukey’s multiple comparison test revealed that NK2571 did not differ from wild type (mean difference 2.69 μm, 95% CI –4.283 to 9.663 μm, p=0.750), DG5020 differed from wild type by ~2–5 germ cell diameters (mean difference of 17.94 μm, 95% CI 10.53 to 25.36 μm, p<0.0001), and DG5131 dramatically differed from wild type (mean difference of 30.36 μm, 95% CI 23.21 to 37.51 μm, p<0.0001). The proliferative zone length of DG5131 was also significantly different from both of its parent strains (NK2571 vs. DG5131 mean difference of 27.67 μm, 95% CI 19.99 to 35.35 μm, p<0.0001; DG5020 vs. DG5131 mean difference of 12.42 μm, 95% CI 4.331 to 20.50 μm, p=0.0006). (E) Plots of length of entire gonad from tip to vulva. N2 (N=12), NK2571 (N=17), DG5020 (N=19), DG5131 (N=20). A one-way ANOVA to determine the effect of genotype on gonad length was significant F3,64=6.27, p=0.0009. Tukey’s multiple comparison test revealed that NK2571 did not differ from wild type (mean difference 31.16 μm, 95% CI –32.99 to 95.32 μm, p=0.578), DG5020 also did not differ from wild type (mean difference of 39.42 μm, 95% CI –23.32 to 102.2 μm, p=0.3546), and DG5131 did differ from wild type (mean difference of 95.15 μm, 95% CI 33.02 to 157.3 μm, p=0.0008). All scale bars 10 μm.

Figure 5—source data 1

Measurements used to generate plots of proliferative zone length, gonad length, and number of mitotic figures for Figure 5.

https://cdn.elifesciences.org/articles/75497/elife-75497-fig5-data1-v1.xlsx

We also counted the number of mitotic figures made by dividing cells in metaphase and anaphase in these strains (Figure 5B) and the total length of the gonad from vulva to tip (Figure 5E). Wild-type N2 had an average of 4.6 dividing cells per gonad; NK2571 had an average of 5.1 dividing cells per gonad (these two were not significantly different); DG5020 had an average of 3.4 dividing cells per gonad; DG5131 had an average of 3.2 dividing cells per gonad (these last two strains were significantly different from wild type, see Figure 5B and legend). Gonad lengths were not significantly different between N2 (average length of 670 µm), NK2571 (639 µm), or DG5020 (631 µm) but were significantly shorter in DG5131 (575 µm).

In the end, we find that only the strain combining inx-8(qy78) and lim-7p::ced-1::GFP has a dramatically smaller gonad that differs from the wild type in three key measures. Expression of the qy78 allele alone with a DTC marker, on the other hand, does not cause any of these quantitative gonad phenotypes. The moderate brood size defects shown by all strains could be caused by numerous processes outside of stem cell regulation. For example, we find the hypothesis of Tolkin et al., 2022, based on the findings of Starich et al., 2020 and Starich et al., 2014, that a major role of inx-8/9 is in the proximal gonad regulating the provisioning of oocytes with essential metabolites, to be compelling. This hypothesis also has support from the large number of unhatched eggs observed for inx-9(qy79[GFP::inx-9]). Thus, we conclude with the observation that endogenous, fluorescently tagged sheath membrane proteins consistently mark both of the distal Sh1 cells without measurably impairing distal gonad function and should be the reagents of choice for live imaging in this cell type. They also consistently report a distal Sh1 position adjacent to the stem cell zone, as we previously found (Gordon et al., 2020).

Discussion

We discovered that the distal position of Sh1 is much closer to the distal end of the young adult hermaphrodite gonad than than was previously observed, where it forms an interface with the DTC’s proximal projections and overlaps substantially with the proliferative zone of the germline where mitotic cell divisions occur (Gordon et al., 2020). Importantly, that study did not claim that Sh1 is necessary or sufficient for what we term ‘niche exit’; we simply observed that Sh1 associates with germ cells as they exit the niche by division. We have now confirmed this finding with functional manipulations of germ cell cycling and cell fate. We observed a distal Sh1 position in other strains with endogenously tagged sheath cell membrane proteins that act in molecular pathways outside of gap junctional coupling, and in a substantial fraction of traditional transgenic animals expressing lim-7 promoter-driven CED-1::GFP, GFP::CAAX and cytoplasmic GFP (though these strains have high variability in fluorescence intensity and localization). Therefore, we consider the results presented here to be confirmatory of the foundational finding of Gordon et al., 2020, which is that almost all mitotic germ cells in the adult hermaphrodite contact the DTC or Sh1, with a noteworthy population in contact with both. Other recent work provides further evidence of a role for sheath cell contact in promoting adult germ cell proliferation, specifically through modulation of Notch receptor glp-1 expression (Gopal et al., 2020). We focus especially on young adults in these studies (less than 24 hr post mid-L4, see Methods). An important caveat to the work is that the gonad is dynamic, and cell shapes and positions change over time. Indeed, dynamic processes could lead to the surprising difference in position often seen between the two Sh1 cells in a single gonad arm, if one Sh1 cell grows more actively over germ cells as they leave the niche. The high variability of expression levels of an overexpressed lim-7p::ced-1::GFP transgene has allowed for this surprising discovery, though that variability makes it a poor marker of the absolute position of the Sh1 cells, it sometimes causes gonad defects, and it interacts synthetically with qy78 to cause germline defects.

This work was inspired by a recent preprint (Tolkin et al., 2021; biorxiv, version 1). This preprint initially reported a severe brood size and embryonic lethality defect in strains bearing the qy78 allele (just over 100 offspring per animal, with day 1 embryonic lethality of nearly 90%) and hypothesized that abnormal innexin function caused by endogenously tagging INX-8 with mKate could be responsible for both the fertility defect and the novel finding of a distal position of Sh1 abutting the stem cell zone. This indeed would be a serious concern, and we are grateful that other researchers in the community are vigilant and interested in the strains we generated.

Tolkin et al., 2022 (revised) downscales concerns about brood size (over 200 offspring per animal) and embryonic lethality (under 10% total) but still proposes that the distal Sh1 position revealed by qy78 is an artifact of protein tagging. The study uses an overexpressed, GFP-tagged, functional cell death receptor protein as the preferred marker of the sheath (the bcIs39 allele encoding lim-7p::ced-1::GFP), but does not justify why such an element should be assumed to be a robust and non-phenotypic marker of the distal sheath. This genetic element—which we demonstrated to interact synthetically with qy78 to cause gonad and germline defects (Figure 5)is present in the genetic background of the worms analyzed in the vast majority of the experiments reported in the four data figures of Tolkin et al., 2022: Figure 1 (all, no true wild-type analyzed), Figure 2 (half of panels D–F with another marker in the other half of those panels, see next), Figure 3 (all, no wild-type analyzed), Figure 4 (all, no wild-type analyzed). In all of these experiments, we conclude the synthetic interaction is driving the phenotypes attributed to qy78, including the dosage-dependent and deletion-mediated suppression of the phenotypes attributed to qy78 shown in Tolkin et al., 2022 (Figure 2—figure supplement 2).

In the rest of Figure 2D-F, Tolkin et al. use a fasn-1::GFP sheath marker coexpressed with qy78. Only six samples were observed, and only 2/6 had the ‘Class 3’ phenotype with a dramatically distal Sh1 border. This differs notably from what is observed for the innexin-defective inx-14(ag17) hypomorphic allele shown in Figure 1—figure supplement 1C for which 21/21 fasn-1::GFP coexpressing samples had ‘Class 3’ gonads. We do not find this experiment to be decisive because of low sample size for this genotype and results in Figure 2F that appear to fall within the distribution of the control.

Next, we consider the other experiments that do not include the bcIs39 marker. In Figure 2B–C and Figure 2–figure supplements 1 and 2, Tolkin et al. use an anti-INX-8 antibody in an immunofluorescence experiment that reports a more proximal Sh1 boundary in the wild type than in worms carrying the qy78 allele. We note the conspicuous absence of innexin detected in the DTC, where it had been reported previously (see the wild-type gonad image in both Figure 3 of Starich et al., 2014, and Figure 2A of Starich et al., 2020). We also note that the anti-INX-8 sheath localization is like a honeycomb, not sparse and punctate. Starich et al., 2020 present evidence that a honeycomb localization pattern for innexins indicates that gap junctions are not forming properly, so this pattern in the wild-type sample is unexpected. Different imaging modalities often yield different patterns, and the previously published control image was made on a compound microscope while the image in Tolkin et al., 2022 (Figure 2B) was made with a confocal microscope. However, it does not seem likely that a switch in imaging approaches would detect more abundant signal in one region (Sh1) and yet lose signal in another (the DTC) in the same field of view. Setting aside questions of reproducibility of this antibody staining experiment, this type of data could shed light on the position of the sheath in a genetically wild-type animal. Of note, Figure 3C of Starich et al., 2014 shows antibody staining of gap junctions forming in what appears to be the Sh1 region at a distance of ~10 germ cell diameters from the distal end.

Transmission electron microscopy was also included in the revised version of Tolkin et al., 2022. The 3D reconstruction in Figure 1—figure supplement 2 shows a sheath that has a honeycomb pattern, presumably due to the thinness of Sh1 as it overlies germ cell bodies (annotation was based on the presence of mitochondria), so we know this technique under-annotates the thinnest regions of the Sh1 cell. It also appears to show what Tolkin et al., 2022, would call a ‘Class 2’ gonad—the DTC and Sh1 cells terminate within ~2 germ cell diameters of one another. It is not representative of a ‘Class 1’ gonad with a large gap (that ‘exceeds 25 µm’). According to the scaling in Figure 1—figure supplement 2, the distal Sh1 annotations fall ~40 µm from the distal end. Taken at face value, this TEM data is equivocal on the question of whether there is a ‘bare region’ between the DTC and Sh1.

When we inspect the TEM stack in Video 4, we observe unannotated structures surrounding the germ cells. They have low complexity, making us wonder if they are an artifact of tissue shrinkage during fixation. Some appear to be contiguous with the annotated somatic cells (DTC and Sh1). Perhaps a more generous annotation would reveal more of the thin somatic cell structures that we know from live imaging are there (minimally, continuous Sh1 cover across germ cell bodies in the more proximal region). With that level of annotation, what distal Sh1 structures would appear? This is a very helpful type of data to include, and hopefully future studies of more TEM samples will help decide the issue.

Finally, each study furnishes additional strains with Sh1 fluorescence expression terminating in variously distal or more proximal domains: Tolkin et al. use fasn-1::gfp (endogenously tagged, cytoplasmic), acy-4::gfp (extrachromosomal, membrane localized), and lim-7p::gfp (integrated array, cytoplasmic); we report ina-1::mNG (endogenously tagged, membrane localized), cam-1::mNG (endogenously tagged, membrane localized), and lim-7p::GFP::CAAX (integrated array, membrane localized). The challenge with definitively proving a more proximal boundary of Sh1—after seeing images like those described in our Figures 2H and 3 and its Figure 3—figure supplement 1, Gordon et al., 2020 (Figure 1—figure supplement 1C) , and Tolkin et al., 2022 (Figure 2—figure supplement 3) —is the challenge of proving a negative. When distal expression is not observed, how can one be certain that the whole sheath is labeled? Absence of evidence is not evidence of absence, and the aforementioned images make it clear that some fluorescent markers fail to capture distal Sh1 structures even when the structures are detectable by other means, and even when they brightly label more proximal cells.

Taken together, we suspect that we are seeing more or less the same things but describing them differently. Tolkin et al., 2022 observe a DTC-Sh1 interface in many worms (~30%) expressing their favored sheath marker (the highly variable bcIs39). These ‘Class 2’ gonads have the pattern that we first reported for endogenously tagged sheath proteins (see Gordon et al., 2020, Figure 1B), including ina-1::mNG (an integrin subunit) and cam-1::mNG (a Wnt pathway member) that are not obvious candidates to have dominant antimorphic effects on innexin signaling. Tolkin et al., 2022 indeed identify several genetic backgrounds in which the spatial relationship between the DTC and Sh1 is perturbed, though how the mechanism acts through changes in innexin function, CED-1::GFP overexpression, or both, remains to be seen. We strongly agree that innexins in the gonadal sheath are important for gonad and germline development.

In physics, the observer effect states that it is impossible to observe a system without changing it. In biological imaging in C. elegans, this means that we can either observe wild-type animals that are dead, dissected and/or fixed and coated or stained, or we can observe genetically modified animals that are alive. Some fine, membranous cellular structures do not survive fixation (Gerdes et al., 2013; Kornberg and Roy, 2014). On the other hand, any genomic modification runs the risk of altering an animal’s physiology.

We feel most confident examining endogenously tagged gene products in Sh1 for several reasons. First, proteins expressed at physiological levels are less likely to directly damage a cell vs. overexpressed fluorescent proteins (Kintaka et al., 2016). Second, the ability to cross-reference among strains with different tagged proteins that act in different molecular pathways allows us to use concordant results in reconstructing cell positions; any single marker may or may not localize to the region of interest, but concordant results among independent experiments help construct an accurate picture of the cell. One factor to consider, however, is that not every endogenously expressed protein is likely to localize evenly across all regions of a cell. We would expect in a large cell like Sh1 that interacts with germ cells in many stages of maturation that some cell-surface proteins would be regionalized. Along those lines, it seems possible that the Sh1 cells might have mechanisms to exclude the cell death receptor CED-1 from the cell membrane domain that contacts proliferating germ cells. The bcIs39 transgene is typically used to study engulfment of apoptotic germ cell corpses at the bend of the gonad and rescues ced-1 loss-of-function mutants for apoptotic germ cell corpse engulfment (Zhou et al., 2001). 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., 2022) 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 need for caution when observing and interpreting endogenously tagged fluorescent proteins is noted. Several steps can and should be taken to increase confidence that a tagged protein is not causing cryptic or unwanted phenotypes. First, multiple edited lines should be recovered and outcrossed, thereby reducing the likelihood that a phenotype is caused by off-target Cas9 cutting creating lesions in any individual edited genome. Second, brood size should be estimated either by timed food depletion (less rigorous) or formal brood size assays (more rigorous). Third, edited lines should be examined for known phenotypes caused by loss of function of the targeted genes. This can be done, in order of least to most rigorous, by consulting the literature, by comparing to RNAi treatments or known mutants, and finally by introducing AID tags and using the degron strategy to deplete the gene product under the lab’s exact experimental conditions of choice (Zhang et al., 2015), however this step will not work for extracellular tags (because extracellular AID tags are not accessible to TIR1 ubiquitin ligase). Finally, any ‘markers’ used should be assessed on their own for phenotypes. Even with these controls in place, synthetic interactions can emerge between ‘markers’ and alleles, including tagged proteins of interest. These interactions can themselves reveal biologically relevant phenomena, but only if they are recognized.

In the end, no transgenic or genome-edited strain is truly wild type, and it should be our expectation that such strains might be somewhat sensitized as a result. Indeed, the synthetic interaction we document between lim-7p::ced-1::gfp and inx-8(qy78) suggests that the qy78 is sensitized for gonad defects caused by other genetic elements affecting the gonadal sheath. However, the perfect reagent does not exit. We can only look for congruent results among a set of independent reagents with non-overlapping weaknesses. Finally, we can formulate questions narrowly enough that, despite their shortcomings, our imperfect reagents are adequate to help answer them. In the future, new endogenously tagged alleles that are expressed in the sheath, single-copy, membrane-localized transgenes that do not affect distal gonad patterning, and different imaging modalities like electron microscopy will shed more light on the complex relationship between the gonadal sheath and the germline. At the present time, however, we consider the existence of an interface between the DTC and Sh1 cells that coincides with the boundary of the distal-most stem-like germ cells to be supported by the preponderance of evidence.

Methods

Strains

In strain descriptions, we designate linkage to a promoter with a p following the gene name and designate promoter fusions and in-frame fusions with a double semicolon (::). Some integrated strains (xxIs designation) may still contain for example the unc-119(ed4) mutation and/or the unc-119 rescue transgene in their genetic background, but these are not listed in the strain description for the sake of concision, nor are most transgene 3’ UTR sequences.

Staging of animals for comparisons among sheath markers

We focused on young adult animals around the time egg laying commences, as in Gordon et al., 2020. Mid L4 animals are picked from healthy, unstarved populations (which are maintained without starving for the duration of the experiment). These animals are kept at 20°C for 16–18 hr, until adulthood is reached and ovulation begins. We prefer not to age the animals much farther into adulthood for routine imaging (though we did this for the temperature shift experiments to follow previously published experimental regimes), as once a full row of embryos is present in the uterus, the distal gonads can become compressed or obscured by embryos. For strains in which a gonad migration defect is observed (DG5020, DG5131), picking animals in the L4 stage prevents bias for or against normal-looking adults (as the defects are profound enough to be visible on the dissecting scope in adults).

Temperature-sensitive mutant analysis

Worms from the emb-30(tn377) mutant genotype were grown at the permissive temperature (16°C) for 24 hr past L4. Plates were shifted to the restrictive temperature (25°C) for 15 hr before DAPI staining, while permissive temperature controls were maintained at 16°C for 18 hr before staining (because development is proportionally slower at 16°C than at 25°C, permissive temperature controls were cultured longer). Two replicates of this experiment were performed with the results combined in Figure 1E. A starting point control (as in Cinquin et al., 2010) and a 21 hr control were also performed, with congruent results (Figure 1—figure supplement 1).

Worms from the glp-1(bn18) mutant genotype were grown at the permissive temperature of 16°C for 24 hr past L4. Plates were shifted to the restrictive temperature (25°C) for 6 hr (Fox and Schedl, 2015). Permissive temperature controls were maintained at 16°C for 6 hr. Worms were imaged live (see Confocal imaging, below).

DAPI staining

DAPI staining was done by modifying standard protocols (Francis and Nayack, 2000), with the cold methanol fixation done for a shorter time (2.5 min) and the concentration of DAPI higher at 1 µg/ml in 0.01% Tween in PBS in the dark for 5 min, washed once with 0.1% Tween in PBS. Samples were briefly stored at 4°C in 75% glycerol and imaged directly in glycerol solution.

Confocal imaging

All images were acquired on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a ×63 Plan-Apochromat (1.4 NA) objective and an ORCAFusion GenIII sCMOS camera (Hamamatsu Photonics) controlled by microManager (Edelstein et al., 2010). RFPs were excited with a 555 nm laser, GFPs were excited with a 488 nm laser, and DAPI was excited with a 405 nm laser. Worms were mounted on agar pads with 0.01 M sodium azide (live) or in 75% glycerol (DAPI stained).

Fluorescence intensity of lim-7p::CED-1::GFP and mKate::INX-8

For quantitative comparisons of fluorescence intensity shown in Figure 3 and Figure 4, gonads were imaged with uniform laser power and exposure times with 1 µm Z-steps. Images were opened in FIJI (Schindelin et al., 2012) and z-projections were made through the depth of the superficial half of the gonad (not including signal from the deep Sh1 cell if it was present). Images without any detectable Sh1 expression were discarded (2/32 images from the analysis in Figure 3A). A line ~20 µm long parallel to long axis of the gonad, terminating near the distal boundary of GFP expression, and not crossing any gaps in Sh1 revealing background was drawn, and average fluorescence intensity was measured along its length in arbitrary units.

Measurements of DTC and Sh1 positions

The distal tip of the gonad was identified in the fluorescence images if the DTC was marked or in a DIC image if the DTC was not marked in a given strain. The distance from the gonad tip to the longest DTC process (when marked), and from the gonad tip to the most distal extent of Sh1 was measured in FIJI (Schindelin et al., 2012). A DTC-Sh1 interface is detected by subtracting the first value from the second value—negative numbers reflect the amount of overlap of these cellular domains across the germline, positive numbers reflect a gap. This is a conservative estimate, as a gap of less than 1 germ cell diameter (~5 µm) would still allow germ cells to contact both the DTC and Sh1 at the same time. Min/max settings on the fluorescence images are adjusted to allow the faintest signal to be detected when measuring.

Analysis of mosaic expression

The variability of the lim-7p::ced-1::gfp transgene allowed us to distinguish the two Sh1 cells in a pair, especially when coexpressed with qy78[mKate::inx-8]. For this experiment, we imaged animals through the full thickness of the distal gonad (40 µm instead of our usual 20 µm that captures just the superficial half of the gonad that can be imaged more clearly). Animals in which two distinct Sh1 cells had different levels of GFP signal were analyzed further for relative cell position. For DG5131, this was 6/19 samples. For DG5020, this was 31/53 samples.

Brood size assays

DG5020 and DG5131 were shipped overnight on 9/23, passaged off the starved shipment plate onto fresh NGM+OP50 plates and maintained by passaging unstarved animals for three generations before beginning the brood size assay. For each strain, 10–15 L4 animals were singled onto NGM plates seeded with OP50 and kept in the same incubator, on the same shelf, at 20°C. The singled animals were passaged once per day on each of the following 5 days to fresh plates, with all plates maintained at 20°C. Two days after removing the parent, the plates with larval offspring were moved to 4°C for 20 min to cause worm motion to cease, and all larvae (and unhatched eggs when noted) were counted on a dissecting scope with a clicker by the same team of worm counters, with internal controls. Plates with unhatched eggs were examined and recounted 1 day later to see if any hatched. Offspring from parent worms that died or burrowed in the process were not counted. Total sample sizes and results reported in Table 1. Replicate brood sizes for DG5020 and NK2571 were performed by a neighboring lab (Dr R Dowen) with the strain names anonymized (Appendix 1—table 1).

Distal germline patterning and mitotic figures

Measurements were made in FIJI from the distal end of the gonad to the transition zone, which is the distal-most row of germ cells with more than one crescent-shaped nucleus. Mitotic figures were counted manually as metaphase or anaphase DAPI bodies. Observations of 0 mitotic figures were counted in the analysis. For Figure 1—figure supplement 1E, measurements were made by manually counting cell diameters. In the distal-most region of the restrictive temperature samples, germ cell nuclei are abnormal, so absolute distances in microns were divided by the diameter of a normal-looking germ cell from the distal end to calculate germ cell diameters in this region.

Gonad length measurements

Strains were synchronized by bleaching and L1 larvae transferred to OP50 seeded NGM plates. At 16°C for 48 hr. L4 worms were picked to fresh plates and cultured at 16°C for an additional 24 hr. These Day 1 adult worms were mounted on agar pads with 0.01 M sodium azide and imaged live. Images were analyzed in FIJI using the segmented line tool from vulva to distal gonad tip (usually in two tiled images to cover the whole gonad length).

Statistical analyses

Tests, test statistics, and p values given for each analysis in the accompanying figure legends. One-way ANOVA followed by Tukey’s multiple comparisons test were conducted in R (R Development Core Team, 2020) or Prism (GraphPad Prism version 9.20 (283) for macOS), GraphPad Software, San Diego, CA.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Caenorhabditis elegans)inx-8https://wormbase.org/Sequence CELE_ZK792.2Encodes gap junction hemichannel subunit
Gene (Caenorhabditis elegans)inx-9https://wormbase.org/Sequence CELE_ZK792.3Encodes gap junction hemichannel subunit
Gene (Caenorhabditis elegans)glp-1https://wormbase.org/Sequence CELE_F02A9.6Encodes Notch receptor
Gene (Caenorhabditis elegans)emb-30https://wormbase.org/Sequence CELE_F54C8.3Encodes putative member of APC
Gene (Caenorhabditis elegans)ina-1https://wormbase.org/Sequence CELE_F54G8.3Encodes worm alpha integrin ortholog
Gene (Caenorhabditis elegans)cam-1https://wormbase.org/Sequence CELE_C01G6.8Encodes worm Wnt receptor
Gene (Caenorhabditis elegans)ced-1https://wormbase.org/Sequence CELE_Y47H9C.4Encodes worm cell death receptor
Genetic reagent (Caenorhabditis elegans)inx-8(qy78(mKate::inx-8)) IV; cpIs122(lag-2p::mNeonGreen:: PLCdPH)Gordon et al., 2020NK2571Can be obtained from K Gordon lab
Genetic reagent (Caenorhabditis elegans)inx-9(qy79(GFP::inx-9)) IV; naSi2(mex-5p::H2B::mCherry::nos-2 3′UTR) IInasi2 transgene from Roy et al., 2018; qy79 from Gordon et al., 2020KLG019Can be obtained from K Gordon lab
Genetic reagent (Caenorhabditis elegans)rlmIs5[lim-7p::GFP::CAAX]This studyKLG020Can be obtained from K Gordon lab
Genetic reagent (Caenorhabditis elegans)qy78(mKate::inx-8) IVThis studyKLG021×2 outcross of NK2571 to N2
Genetic reagent (Caenorhabditis elegans)inx-9(qy79(GFP::inx-9)) IV; naSi2(mex-5p::H2B::mCherry::nos-2 3′UTR) II; glp-1(bn18) IIIglp-1(bn18) from Kodoyianni et al., 1992 doi: 10.1091/mbc.3.11.1199KLG022Mutant obtained from CGC, crossed to KLG019
Genetic reagent (Caenorhabditis elegans)inx-8(qy78(mKate::inx-8)) IV; cpIs122(lag-2p::mNeonGreen:: PLCdPH); emb-30(tn377) IIIemb-30(tn377) from Cinquin et al., 2010 doi: 10.1073/pnas.0912704107KLG023Mutant obtained from CGC, crossed to NK2571
Genetic reagent (Caenorhabditis elegans)cp243[cam-1::mNG]Heppert et al., 2018 doi:10.1534/GENETICS.117.300487LP530Can be obtained from B Goldstein lab
Genetic reagent (Caenorhabditis elegans)qy23[ina-1::mNG]Jayadev et al., 2019 doi: 10.1083/jcb.201903124NK2324Can be obtained from D Sherwood lab
Genetic reagent (Caenorhabditis elegans)tnIs6[lim-7p::GFP]Hall et al., 1999DG1575Obtained from CGC
Genetic reagent (Caenorhabditis elegans)bcIs39[lim-7p::ced-1::GFP];naIs37Tolkin et al., 2022; naIs37 originally from Pekar et al., 2017DG5020See Tolkin et al., 2022
Genetic reagent (Caenorhabditis elegans)qy78[mKate::inx-8];bcIs39[lim-7p::ced-1::GFP];naIs37Tolkin et al., 2022DG5131See Tolkin et al., 2022
Software, algorithmμManager software v1.4.18(Edelstein et al., 2010) doi: 10.1002/0471142727.mb1420s92RRID:SCR_016865https://micro-manager.org/
Software, algorithmFIJI 2.0Schindelin et al., 2012 doi: 10.1038/nmeth.2019RRID:SCR_002285https://fiji.sc/
Software, algorithmGraphPad Prism version 9.20 (283) for macOSGraphPad Software, San Diego, CARRID:SCR_002798https://www.graphpad.com/
Software, algorithmAdobe Illustrator CCAdobe Systems IncRRID:SCR_010279
Appendix 1—table 1
Replicated, anonymized brood size assay (Dowen Lab).
StrainTolkin (v1)Tolkin (revised)Li (submitted)Dowen (anon.)Li vs. Dowen
NK2571 (qy78; cpIs122)155±24.4 (n=19)212.8±27.5 (n=23)220±41 (n=15)233±32 (n=11)t=0.899, p=0.378, n.s.
DG5020 (lim-7p::ced-1::GFP; naIs37)235.8±43.2 (n=56)237.5±46.5 (n=24)202±29 (n=12)213±52 (n=12)t=0.643, p=0.527, n.s.

Data availability

Source data files contain the numerical data used to generate the figures.

References

    1. Evans TC
    (2006)
    The C. Elegans Research Community, WormBook
    Transformation and microinjection, elegans research community, wormbook, The C. Elegans Research Community, WormBook, Wormbook, 10.1895/wormbook.1.108.1.
  1. Software
    1. R Development Core Team
    (2020) R: A language and environment for statistical computing
    R Foundation for Statistical Computing, Vienna, Austria.

Decision letter

  1. Yukiko M Yamashita
    Reviewing Editor; Whitehead Institute/MIT, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands
  3. Yukiko M Yamashita
    Reviewer; Whitehead Institute/MIT, United States
  4. Judith Yanowitz
    Reviewer; Magee-Womens Research Institute/ U. Pittsburgh School of Medicine, United States
  5. Ekaterina Voronina
    Reviewer; University of Montana, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including X as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Judith Yanowitz (Reviewer #2); Ekaterina Voronina (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The reviewers agreed that the manuscript represents an important advance that is based on the previous eLife paper (Gordon et al.). However, there are important issues brought up by reviewers that need to be addressed as you can see below.

Reviewer #1:

Overall, it seems that this manuscript presents a compelling case that their earlier conclusion was correct. However, the data as well as explanation are sometimes not detailed enough, making it difficult to assess the rigor of the work. As detailed below, I'd like to see a bit more data that affirm their conclusion.

– line 62-69 is very difficult to understand. The emb-30 mutant and glp-1 mutant are not explained sufficiently (what they are, what phenotypes to expect in more details are needed), making it hard to understand the flow of the logic. The results seem to be consistent with the notion that Sh1 influences the differentiation of germ cells, once they exit the stem cell niche created by the DTC. But I think at least more thorough explanation is necessary, and perhaps additional more convincing data are needed. In Figure 1, 'germ cell phenotype' is only assessed by DAPI staining (the other markers only telling the location of DTC and Sh1) and it cannot be concluded for sure whether their assessment is correct.

– Fig4E (legend says it's G, please correct) - what it tries to show is unclear to non-expert. Please add control and elaborate exactly what is a defect.

– Figure 5: this is a critical figure to prove that Tolkin et al. might have used a combination of transgenic alleles that together causes germ cell defects. However, the 'phenotypes' is only assessed by DAPI in Figure 5, and it is unclear whether their observations (defects, or lack thereof) are comparable to what Tolkin et al. described in their manuscript.

Reviewer #2:

The Li et al. paper is essentially a response to recently submitted manuscript, Tolkin et al. 2021 and a follow-up on the senior author's prior work characterizing the relationship between Sh1, the DTC, and germ cell proliferation. Overall the manuscript provides evidence that the ced-1::GFP marker in Sh1 appears to be deleterious and therefore argues for caution when using extrachromosomal arrays and when relying heavily on single reporters to assess cell morphology. However, there is little new in the data, but rather it stands as a commentary to Tolkin et al.

One of the critical issues here as that the Tolkin paper suggests that the tagged inx-8 allele should not be used as a Sh1 marker since it is dominant negative. This paper suggests that there are defects associated with lim-7::CED-1::GFP which preclude this as a marker. While substantive data supports the latter (albeit with fairly low N numbers), the authors do not address whether there might be problems with the inx-8 mKate strain. They do, however, argue about the validity of their observations by a detailed analysis of imaging data that accounts for weak fluorescence and by use of multiple difference promoter GFP transgenes.

One of the major areas of conflict between this paper and Tolkin et al. is whether the inx-8 tagged strains has brood size defects: this paper and prior work say no; the Tolkin paper says yes. Are there differences in the growth media or temperatures used in the different labs? This should be resolved between the labs.

There is some concern that a subset of the markers are extracellular vs intracellular and the extracellular markers could be cleaved – an issue that is dismissed.

For all figures, the transgenes/tagged proteins used and specific strains must be listed in the figure and legend.

The statement that "A recent study (Tolkin et al., 2021) uses this transgene in all 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." Is untrue. The Tolkin paper also used Plim-7::GFP without CED-1. Please modify this statement and clarify how you think that marker would be incorrectly analyzed.

It is very difficult to interpret the images in Figure 1 and 2: in some cases the DTC has long projection, in others it does not; in some images the germ lines are fat (Figure 1A,B) or bent and deformed (Figure 2A,C). IN figure 2F, it is unclear why this is DIC +GFP since the full GFP signal may not be apparent with the DIC and why are there lots of eggs around. Overall an improvement in the imaging would better help the reader understand the data and compare across figures/genotypes.

The description of the emb-30 and glp-1 alleles is confusing. It is not clear why these alleles would be a test of their hypothesis. Also of concern is that the shift of these alleles is done is L4/adulthood after the Sh1 cells is differentiated? Are they arguing that the Sh1 does not change in morphology in response to the shift in the germ cell state?

In Figure 2A-C, an asterick should be used to mark the DTC.

In text it would be helpful to better describe what Figure 1 is showing. What do you mean by "pink arrow marking gc transition".

The actual distance here is irrelevant if the germ lines are different sizes. A better measure is the number of nuclear widths.

In the methods, it is stated that, 16{degree sign}C experiments are cultured longer (for emb-30), but what is the evidence to support the choice of a 3 hour difference? Why is a similar criterion not use for the glp-1(ts)?

DAPI staining in the methods: "DAPI staining was done according to standard protocols". Needs to be referenced. I am shocked at the concentration of DAPI. My understanding is that concentrations of 1/50,000 dilution of 10ug/ml stock are used. Was this really not in water and not buffer?

While the desire to be concise is appreciate, in the methods or an accompanying table, the full description of the strains must be provided.

There needs to be clarification as to the source of the strains. (perhaps in a strain table).

What is meant by "DG5020 and DG5131 were shipped overnight on 9/23". Shipped from where?

In table 1, subscript "b" says: "N2 numbers come from multiple trials, not all of which counted negligible numbers of dead embryos, including the trial in which ina-1(qy23) and cam-1(cp243) were counted". Does this mean that in a subset of the trials, there was embryonic lethality in the N2 strain? How is the accounted for? Why is it not shown?

Also related table 1, it is stated: "our endogenously tagged inx-8(qy78) and inx-9(qy79) strains had dramatically different degrees of embryonic lethality, with qy79 producing over 150 unhatched eggs per worm." When I look at the table, the brood size for inx-9 is ~226 with 41% embryonic lethality. This would lead to on average ~85 unhatched eggs/worm not 150. Where do these numbers come from and which is correct?

Given that there are differences between the labs in the behaviors of different strains, it is critical that Kacy et al. perform the control brood sizes on the inx-8 and inx-9 described in lines 231 -236 since a critical aspect of their interpretation hinges on this.

Lines 239-240, please provide references for other brood sizes conducted in the lab.

What do you mean in Figure 4 by aberrant engulfment? It seems it is just pointing to foci at the cell membranes that could be junctions between the cells.

Lines 114-121 should refer to an image figure (3C,D)

Reviewer #3:

In this manuscript, Li et al. describe their further investigation of somatic Sh1 cells association with underlying germ cells. This work provides further support for the model where the distal border of Sh2 cell is forming an interface with DTC and Sh1 overlays the mitotic germ cells poised for differentiation. These findings confirm and extend the previous report, but do not test the functional significance of Sh1/germ cell association. Additionally, through the analysis of coexpressing CED-1::GFP and mKate::INX-8, this work revealed that two Sh1 cells may have drastically different levels of CED-1::GFP expression, while maintaining relatively similar mKate::INX-8 levels. The distinct regulation of transgene expression and asymmetric architecture of Sh1 cells around the germ cells have not been appreciated before, but the functional significance of these findings is unclear. The authors argue that overexpression of CED-1::GFP is detrimental as it is associated with gonad morphology defects, and further propose that high levels of CED-1::GFP expression in distal Sh1 are not well-tolerated.

Overall, this manuscript presents findings that confirm the previously published model; however, several discrepancies between this manuscript and Tolkin et al. preprint are concerning:

1) bcIs39 CED-1::GFP transgene was reported to perfectly overlap with mKate::INX-8 in >85% of cases by Tolkin et al., but only in 60% of cases in the current work.

2) The reported brood size and embryonic lethality for several identical or equivalent strains are vastly different. Specifically, NK2571 inx-8(qy78) brood size is 220 vs 155 with embryonic lethality of 8% vs 58% and DG505 embryonic lethality is 20% vs 1%. The causes for these differences need to be identified (culture conditions? temperature? formulation of culture plates?); otherwise these problems will persist/emerge for any other research group using these markers leading to issues with result reproducibility. In contrast to the assertion in Line 243, Tolkin et al. have assessed qy78 allele for brood size and embryonic lethality by itself after an outcross or in the NK2571 strain containing cpIs122 transgene, not in combination with bcIs39. Therefore, one cannot argue that brood size and embryonic lethality defects originated from the genetic interaction of qy78 and bcIs39.

Additionally, a detailed analysis of CED-1::GFP marker of Sh1 in Figure 4 revealed that ~50% of morphologically-normal gonads display an interface between DTC and Sh1, while remaining morphologically-normal gonads show a gap between these cells. This provides an opportunity to test the assertion of the model put forth by Gordon et al. 2020 and challenged by Tolkin et al. preprint – that the distal boundary of Sh1 cells impacts germ cell switch from proliferation to differentiation. According to Gordon et al., 2020 model, the proximal displacement of Sh1 in 50% of gonads expressing CED-1::GFP is expected to shift the position of meiotic entry away from the distal end resulting in a larger distal mitotic region in these germlines. By contrast, data in Figure 5 shows a shorter mitotic region in both strains expressing CED-1::GFP, consistent with Tolkin et al's conclusion that Sh1 position does not affect meiotic entry. Therefore, it appears that while the normal position of Sh1 distal boundary is closer to DTC than previously appreciated, its displacement is unlikely to affect the underlying germ cell population.

Other suggested revisions:

1. Line 180: the conclusion that bcIs39 "sensitizes worms for gonad morphology defects" is unwarranted as disruption of DTC migration appears similar in both described genetic backgrounds. It appears that bcIs39 directly disrupts DTC migration.

2. Line 196: remove "of".

3. Line 209: using CRISPR/Cas9 *to* introduce… (add "to").

4. Figure 1 and legend: include the allele designations of edited inx-8 and inx-9 for consistency with other figures.

5. Figure 1D: The position of Sh1 distal boundary in the right column (restrictive temperature) is hard to judge; the dashed line indicates a distal projection in the middle that is not apparent by diffuse GFP signal.

6. line 546, 557, 564 and Figure 1E: I don't think "gc transition" is an accepted term in the field. Perhaps replace with "mitotic cell population boundary"?

7. line 571: the legend indicates strain ID only for DG5020; is this necessary? If so, all strain IDs need to be included.

8. line 599: change panel to (D).

9. line 609: change panel to (E).

https://doi.org/10.7554/eLife.75497.sa1

Author response

Reviewer #1:

Overall, it seems that this manuscript presents a compelling case that their earlier conclusion was correct. However, the data as well as explanation are sometimes not detailed enough, making it difficult to assess the rigor of the work. As detailed below, I'd like to see a bit more data that affirm their conclusion.

-line 62-69 is very difficult to understand. The emb-30 mutant and glp-1 mutant are not explained sufficiently (what they are, what phenotypes to expect in more details are needed), making it hard to understand the flow of the logic. The results seem to be consistent with the notion that Sh1 influences the differentiation of germ cells, once they exit the stem cell niche created by the DTC. But I think at least more thorough explanation is necessary, and perhaps additional more convincing data are needed. In Figure 1, 'germ cell phenotype' is only assessed by DAPI staining (the other markers only telling the location of DTC and Sh1) and it cannot be concluded for sure whether their assessment is correct.

Thank you very much for pointing out these experiments are not described sufficiently well. We have expanded the description of what these temperature sensitive alleles have been used for in the past, how our results agree with these prior findings, and furthermore how our result reveal the position of the sheath relative to the stem-like population of germ cells. Pages 1-3 have a rewritten section describing these experiments.

This assay is not demonstrating that Sh1 influences the germ cells. Instead, the original experiments helped to identify a feature of the germline—its division into a distal, stem like population and a more proximal, mitotically proliferating population that is fated to differentiate though has not yet entered the meiotic cell cycle. We find that the distal position of Sh1 falls at the same proximodistal position as the boundary between stem and non-stem germ cells, and it is the cells beneath Sh1 that differentiate first when progress through the cell cycle is halted or the germ cell receptor of the stemness cue is deactivated. This evidence independently supports the conclusion we drew from the experiments reported in Gordon et al., 2020, Figure 5.

While we are eager to share the results of an experiment that builds of off foundational work in the field to confirm our 2020 findings, we did these experiments independently of and prior to our engagement with the Tolkin et al. group, and they are not meant to address that study directly. If our paper ends up published as a direct response to Tolkin et al., we are open to removing Figure 1 to keep the study focused. We appreciate your guidance on this subject.

– Fig4E (legend says it's G, please correct) - what it tries to show is unclear to non-expert. Please add control and elaborate exactly what is a defect.

Thank you for pointing this out; we agree that this figure needs more explanation (and a correct letter in the legend!). The lim-7p::CED-1::GFP transgene is typically used to visualize the engulfment of apoptotic germ cells near the bend of the gonad; this region of the germline undergoes physiological apoptosis, in which dying germ cells give cytoplasm to their sisters to inflate oocytes. The CED-1::GFP signal on the sheath membrane forms concentrated bubbles around germ cells being engulfed. We observe such “bubbles” of GFP in some samples of DG5131 (2/18 in the relevant dataset) that are located abnormally distal in the gonad (2 projections of the same gonad from Figure 4E). There are so many abnormal Sh1 membrane protrusions that we agree with the reviewer that the full projection is difficult to understand. This isn’t a phenotype we want to characterize, but it provides further evidence that this strain has abnormal gonad biology.

We have updated the projection shown in in Figure 4E and labeled the engulfed germ cells.

– Figure 5: this is a critical figure to prove that Tolkin et al. might have used a combination of transgenic alleles that together causes germ cell defects. However, the 'phenotypes' is only assessed by DAPI in Figure 5, and it is unclear whether their observations (defects, or lack thereof) are comparable to what Tolkin et al. described in their manuscript.

Thank you for the helpful suggestion to compare our results directly with the measurements reported by Tolkin et al. The most relevant figure from Tolkin et al. to the results shown in our Figure 5 is the experiment in Tolkin Figure 4, in which fixed, dissected gonads of various genotypes were antibody stained for the CYE-1(+) progenitor pool and anti-GFP against the lim-7p::ced-1::GFP transgenic protein that they use to label the sheath. The CYE-1(+) technique (from Mohammad et al., 2018) reveals a proximal boundary of CYE-1(+) cells that falls a bit distal to the crescent shaped nuclei of the “Transition Zone”. Therefore, while we don’t expect the PZ measurements in each dataset to be an exact match (ours should be longer), we do expect the magnitude and direction of difference between strains to be concordant.

We can use Mohammed et al. (2018) Figure 2 for the wild-type position of a CYE-1(+) domain. The Tolkin analysis lacks a wild-type control and does not analyze the qy78 allele on its own without bcIs39(lim-7p::ced-1::gfp) in the background (“NA” in Author response table 1). Also, without the raw data, I must infer the mean measures from the display items in Mohammed Figure 2, Tolkin Figure 4, and its supplement. With those caveats, this is my best effort to compare between the experiments:

Author response table 1
Strain, GenotypeTolkin Figure 4 labelTolkin CYE-1(+) domainMohammed Figure 2 CYE-1(+) domainMohammed Figure 2 DAPI, est. from 1 sampleLi Figure 5 labelLi DAPI mean measurement of PZ
N2 wild-typeNANA~95 um
~21 gcd
~113 um
~28 gcd
Wild type N2109 +/- 13 um
27 +/- 3 gcd
NK2571 (qy78;cpIs122)NANANANAinx-8(qy78);cpIs122106 +/- 12 um
26 +/- 2 gcd
DG5020 (bcIs39; naIs37)“markers only” (4A)
“Wild-type” (4C)
~70 um
~17 gcd
NANADG5020(bcIs39, naIs37)91 +/- 19 um
23 +/- 3 gcd
DG5131 (qy78;bcIs39; naIs37)inx-8(qy78)~60 um
~15 gcd
NANADG5131(qy78;bcIs39; naIs37)79 +/- 13 um
20 +/- 2 gcd

First, as expected, when both measurements have been made for the same strain, the CYE(+) domain is ~5-6 germ cell diameters or ~20 microns shorter than the DAPI-stained Proliferative Zone. This agrees with the observations of Mohammed et al. (2018). Next, wild-type N2 have the longest proliferative zone, with DG5020 having a somewhat shorter proliferative zone, and DG5131 having a profoundly shorter proliferative zone. In our analysis, we find that NK2571(qy78;cpIs122) has a proliferative zone that is statistically indistinguishable from wild type (Tolkin did not analyze either fully wild-type or NK2571 in this experiment). Importantly, the results shown in Tolkin Figure 4 and its supplement show that DG5020 has an abnormal distal gonad compared to previously published wild-type measurements, which is consistent with our findings.

We have now updated our Results section in Line 281-323 to clarify this comparison. We also have increased the N for all strains analyzed (see Figure 5 legend).

To address the issue that we report only lengths of the proliferative zone using DAPI staining, in this revision, we add two additional measurements: incidence of germ cell division and total gonad length (Figure 5B and 5E). Overall, these tell a consistent story: in the NK2571 strain for which we observe a far distal Sh1 cell boundary, the germline is statistically indistinguishable from wild type. On the other hand, the “markers only” DG5020 strain preferred by Tolkin et al. is slightly diminished in all measures. Finally and crucially, the combination of alleles in the DG5131 strain that Tolkin uses to assess the function of the qy78 allele in nearly all experiments is dramatically impaired both for the size of the proliferative zone, the number of actively dividing germ cells observed, and the length of the gonad. We believe this is strong evidence to refute the idea that qy78 is a “poison” allele on its own; instead, there is a synthetic interaction with the markers used by Tolkin et al.

Reviewer #2:

The Li et al. paper is essentially a response to recently submitted manuscript, Tolkin et al. 2021 and a follow-up on the senior author's prior work characterizing the relationship between Sh1, the DTC, and germ cell proliferation. Overall the manuscript provides evidence that the ced-1::GFP marker in Sh1 appears to be deleterious and therefore argues for caution when using extrachromosomal arrays and when relying heavily on single reporters to assess cell morphology. However, there is little new in the data, but rather it stands as a commentary to Tolkin et al.

One of the critical issues here as that the Tolkin paper suggests that the tagged inx-8 allele should not be used as a Sh1 marker since it is dominant negative. This paper suggests that there are defects associated with lim-7::CED-1::GFP which preclude this as a marker. While substantive data supports the latter (albeit with fairly low N numbers), the authors do not address whether there might be problems with the inx-8 mKate strain.

Thank you for this suggestion to improve our sample sizes and to draw attention to the discussion of the shortcomings of our own fluorescently marked strains.

We have made our discussion of these weaknesses explicit (lines 418-421):

“In the end, no transgenic or genome-edited strain is truly wild type, and it should be our expectation that such strains might be somewhat sensitized as a result. Indeed, the synthetic interaction we document between lim-7p::ced-1::gfp and inx-8(qy78) suggests that the qy78 is sensitized for gonad defects caused by other genetic elements affecting the gonadal sheath.”

We report and discuss the reduced brood sizes of qy78 and other fluorescently marked strains in Table 1 and discussion thereof.

We have also increased our sample sizes (in all figure legends).

They do, however, argue about the validity of their observations by a detailed analysis of imaging data that accounts for weak fluorescence and by use of multiple difference promoter GFP transgenes.

We appreciate that the reviewer found the congruence between different fluorescence patterns to be good evidence for the existence of a distal Sh1 boundary. To clarify, these are not promoter::GFP transgenes in Figure 2A-D, these are CRISPR/Cas9-mediated fusions between endogenous protein coding genes and fluorescent protein coding genes. In addition to the inx-8 and inx-9 tags, we also observe distal Sh1 expression in CRISPR-tagged ina-1 and cam-1 strains. These latter two genes are not innexin genes (one encodes an integrin subunit, the other a Wnt receptor complex member). These proteins localize to the cell membrane.

The fluorescence in Figure 2A-D is dimmer and more punctate because these are tagged worm proteins expressed by their two genomic copies from endogenous promoters, localizing to the cell membranes in particular ways. This makes them less variable and less prone to silencing than overexpressed transgenes. However (related to the prior point about qy78), these tags also may potentially affect the function of the tagged proteins themselves. We consider this risk, and ways to address it, in the Discussion section (lines 381-416). We do not see a way in which tagging integrin or a Wnt receptor could affect innexin hemichannel function in the same way, however, so the distal Sh1 boundary is not only seen in strains with abnormal innexin proteins, as Tolkin et al. propose.

One of the major areas of conflict between this paper and Tolkin et al. is whether the inx-8 tagged strains has brood size defects: this paper and prior work say no; the Tolkin paper says yes. Are there differences in the growth media or temperatures used in the different labs? This should be resolved between the labs.

We agree that this crucial question must be resolved, and it was our priority in revising the manuscript (see first section of this letter). The Tolkin paper has now dropped the brood size defect and embryonic lethality claims.

There is some concern that a subset of the markers are extracellular vs intracellular and the extracellular markers could be cleaved – an issue that is dismissed.

Based on 3D models of another innexin (Oshima et al., 2016), we suspect our N-terminal tags on mKate::INX-8 and GFP::INX-9 are extracellular. We do not address (or explicitly dismiss) potential N-terminal cleavage in this manuscript, but we also don’t suspect it for several reasons. First, the mKate::INX-8 fluorescence is punctate, suggesting that anchoring of some kind rather than free diffusion within the gonad governs its localization. Second, where mKate::INX-8 is coexpressed with a DTC or sheath membrane marker (lag-2p::mNG::PH or lim-7p::CED-1::GFP), we see mKate colocalization at the cell membrane, not diffusion away from the GFP signal. Finally, we see considerable localization of the mKate::INX-8 signal within oocytes and embryos (Gordon et al., 2020, Figure S1), which Starich et al. 2014 report in their antibody staining experiments (that paper explains that the INX-8 protein is internalized from the sheath-germ cell interface, not produced by the embryos or germ cells). While this question of protein processing is interesting, we worry it would be a distraction to include this discussion in the manuscript. Our extracellular, membrane-tethered fluorescent protein tags might circumvent a problem with visualizing the Sh1 cell, which is that its thin, flat shape leaves relatively little room for cytoplasmic fluorescent proteins to accumulate to visible levels, hence the “honeycomb” appearance of cytoplasmic GFP in that cell.

In the revised version of the manuscript, Tolkin et al. add a CRISPR tagged FASN-1::GFP as well, a GFP fusion to an endogenous locus encoding fatty acid synthase (presumably with cytoplasmic localization). They have also added an extrachromosomal array transgene expressing a fusion of ACY-4::GFP, which is predicted to be localized to the membrane, and which has the most “Class 2” gonads with a DTC-Sh1 interface of any of their markers (Figure 1—Figure supplement 1). The same caveats about variability of extrachromosomal arrays apply to this strain; absence of expression should not be construed as absence of a cell.

For all figures, the transgenes/tagged proteins used and specific strains must be listed in the figure and legend.

Thank you for this suggestion. We hope to keep the nomenclature transparent and accessible, but agree with the reviewer that precision is very important and now include full strain information in all figure legends.

The statement that "A recent study (Tolkin et al., 2021) uses this transgene in all 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." Is untrue. The Tolkin paper also used Plim-7::GFP without CED-1. Please modify this statement and clarify how you think that marker would be incorrectly analyzed.

Thank you for pointing out the need to correct “all” to “most” (line 396). Tolkin et al. do indeed use lim-7 promoter-driven cytoplasmic GFP strains in Figure 1—Figure Supplement 1 (where the lim-7p::GFP is coexpressed with a DTC marker in otherwise wild-type and inx-14(ag17) backgrounds) and Figure 2—Figure Supplement 2 (where it is coexpressed with a DTC marker and inx-8(qy78)).

Our results for lim-7::GFP in Li Figure 2F do not agree with the results shown in Tolkin Figure 1—Figure Supplement 1; we find a mean distance of Sh1 from the distal end of 40.63 microns (N=20), Tolkin et al. show a mean of ~80 microns. Of note: this distal Sh1 position is dramatically different from what Tolkin et al. report for the bcIs39[lim-7p::ced-1::gfp] sheath marker strain (~50 microns, same figure). The internal inconsistency between these two markers is not discussed in that study.

We cannot comment further on this discrepancy except to say we measured sheath position in a different strain, strain DG1575 from the CGC (ordered 06-15-2020) which carries the tnIs6 allele and was used to visualize the sheath in Hall et al., 1999. As for how it could be analyzed incorrectly, we suspect the same issue of variable/low levels of GFP expression from the lim-7 promoter in the distal sheath could be at work in this case as well as for the lim-7::ced-1::GFP allele which we discuss at length.

We also analyzed this tnIs6 allele in Gordon et al., 2020 (Figure 1—Figure Supplement 1C) in the presence of the inx-8(qy78) allele, and in that case find results that almost precisely agree with Tolkin’s conclusion in Figure 2—Figure Supplement 2. The two markers largely overlap (in our hands, 11/12 and 24/28 gonads in two separate replicates), and when they don’t, the mKate::INX-8 expression is farther distal than the GFP signal, due to apparent loss of GFP in the distal-most Sh1 cell.

It is very difficult to interpret the images in Figure 1 and 2: in some cases the DTC has long projection, in others it does not; in some images the germ lines are fat (Figure 1A,B) or bent and deformed (Figure 2A,C). IN figure 2F, it is unclear why this is DIC +GFP since the full GFP signal may not be apparent with the DIC and why are there lots of eggs around. Overall, an improvement in the imaging would better help the reader understand the data and compare across figures/genotypes.

Thank you for the suggestions to improve these figures. Asterisks have been added to the distal end of all gonads in every figure.

In Figure 1, we are not visualizing dedicated DTC markers. Fluorescence from the cpIs122 lag-2p::mNG::PH is quenched by methanol fixation for DAPI staining in Figure 1B (hence no DTC outline indicated), while the inx-9(qy79) allele has robust but punctate GFP::INX-9 localization on both Sh1 and DTC (outlined in white, Figure 1D). Additionally, we have now further highlighted that the experiments in Figure 1 depict older animals than the other figures, based on the timing used in the experiments we are replicating. The emb-30 allele certainly causes gonads to look fat at the restrictive temperature; in this strain, germ cells stop moving proximally.

In Figure 2D and 2F, we feel it is important to show the DIC channel so the distal tip of the gonad is pictured. We have taken care not to wash out any GFP signal in the merged image. Eggs are sometimes visible, as the uterus lies immediately ventral to the distal tip of the gonad, and we are looking at reproductive adults in their first day of egg laying. Slight bends at the distal end of gonads are not deformities, but artifacts of coverslipping; the gonad tip is free in the body and when compressed, settles around gut or eggs nearby.

The description of the emb-30 and glp-1 alleles is confusing. It is not clear why these alleles would be a test of their hypothesis. Also of concern is that the shift of these alleles is done is L4/adulthood after the Sh1 cells is differentiated? Are they arguing that the Sh1 does not change in morphology in response to the shift in the germ cell state?

The reviewer is correct that our results show that the position of Sh1 does not change in these temperature sensitive mutants, and that its distal position does not differ from the position of the germ cell population that differentiates first after temperature shift. We have improved our explanation of these experiments thanks to this helpful feedback and the similar suggestions of Reviewer 1 (above). Specifically, we have explained that we are using the germ cell perturbations of the ts alleles to reveal distal (stem-like) and proximal (still mitotic, but more genetically differentiated) germ cell populations, in relation to the position of the Sh1 cells. In both experiments, we find that the pool of germ cells that differentiates first at the restrictive temperature is the pool that lies under the Sh1 cells. This confirms that these cells have left the niche.

In Figure 2A-C, an asterick should be used to mark the DTC.

In text it would be helpful to better describe what Figure 1 is showing, What do you mean by "pink arrow marking gc transition".

The “transition zone” of the germline is the position where the nuclei take on the characteristic crescent shape of leptotene/zygotene of meiosis I. While some of the small, circular nuclei distal to this position are in S phase of meiosis I, the vast majority are mitotic germ cells, so the gonad distal to the transition zone is referred to as the proliferative zone. However, in the temperature-shifted experiments, it seemed wrong to call this the “proliferative zone” because the cells have ceased to proliferate. However, the difference in nuclear morphology can still be observed.

The actual distance here is irrelevant if the germ lines are different sizes. A better measure is the number of nuclear widths.

The results from the emb-30 experiment are difficult to analyze in terms of germ cell diameters, because the germ cell nuclei are malformed due the failure of metaphase-anaphase transition. However, we added this measurement for one of the replicates (shown in Figure 1—Figure supplement 1E).

In the methods, it is stated that, 16{degree sign}C experiments are cultured longer (for emb-30), but what is the evidence to support the choice of a 3 hour difference? Why is a similar criterion not use for the glp-1(ts)?

Thank you for pointing out the need to better explain these experiments, which Reviewer 1 also noted (see above). These experiments were inspired by key prior work that provides evidence for a distal pool of stem-like germ cells; in this paper we repeat these experiments in the presence of our fluorescent DTC and sheath markers to observe the positions of the soma and stem-like germ cell pool. These genetic loss of function experiments provide corroborating evidence that the germ cells that lie under the Sh1 cells are further along the path to differentiation than the distalmost stem-like cells. Our 2020 study used fluorescent markers related to germ cell fate to first test this hypothesis.

The original emb-30(ts) experiment by Cinquin et al. (2010) imaged a starting point control but had no aged control that was kept at the permissive temperature while the experimental group was shifted to the restrictive temperature for 15 h. This seemed like a nonideal control to us since the gonad and germline change over time, so the initial 18 hour control was chosen for experimenter convenience; the experimental group was fixed, stained, and imaged at 15 hours with the control continuing to run in the meantime. We have since repeated the experiment with a starting point control only (as in the original study), and we see the same result that we obtained with the 18 hour control, as well as a 21 hour control we recently performed (Figure 1—Figure supplement 1). This suggests that by 36 hours post mid-L4, the relationship between the stem cell zone and Sh1 cell are fixed.

On the other hand, the original glp-1(bn18) experiments of Fox and Schedl (2015) included controls that were exactly matched for time, so our use of this allele similarly uses a control kept at the permissive temperature for the same amount of time that the experimental group is observed.

DAPI staining in the methods: "DAPI staining was done according to standard protocols". Needs to be referenced. I am shocked at the concentration of DAPI. My understanding is that concentrations of 1/50,000 dilution of 10ug/ml stock are used. Was this really not in water and not buffer?

Thank you for pointing this out! DAPI was reconstituted in distilled water per the MSDS, but you are correct that the working solution was made in 0.01% Tween in PBS. The lower end of suggested working concentrations given on the MSDS is 1 ug/ml. We noted that this is higher than the Nayack Lab protocol suggests, but we get clean staining, even if we waste some dye. Since we cut our methanol fixation short to preserve mKate::INX-8 fluorescence, we probably have lower tissue penetration to compensate for.

While the desire to be concise is appreciate, in the methods or an accompanying table, the full description of the strains must be provided.

Thank you, a Key Resources Table has now been composed. We have also improved Table 1 as suggested (below).

There needs to be clarification as to the source of the strains. (perhaps in a strain table).

What is meant by "DG5020 and DG5131 were shipped overnight on 9/23". Shipped from where?

The full details for each strain now appear in the Key Resources Table.

Perhaps that level of detail on strain history is unnecessary. We wished to express that the strains we imaged for this manuscript were analyzed directly after receipt from the Tolkin et al. collaborators, not after long-term culture (during which strains can theoretically diverge). We understand that this is not a typical part of a strain description, but when we are looking at nominally the same strains and reporting different phenotypes, it seems important. We did however delete references to details of strain maintenance in the Results section.

In table 1, subscript "b" says: "N2 numbers come from multiple trials, not all of which counted negligible numbers of dead embryos, including the trial in which ina-1(qy23) and cam-1(cp243) were counted". Does this mean that in a subset of the trials, there was embryonic lethality in the N2 strain? How is the accounted for? Why is it not shown?

We have not typically quantified embryonic lethality when performing brood size assays, and we always include an N2 wild-type control population. In this case, we ran one brood size assay for strains LP520 and NK2324 with a wild-type N2 control and did not score embryonic lethality. Later, when we received the Tolkin strains, we ran a brood size assay for DG5020 and DG5131 along with our strains NK2571 and KLG019 and an N2 control, and we did score embryonic lethality because we saw that the other group had done so. So while we can pool the N2 brood size, we can’t report a total embryonic lethality for the same dataset.

Also related table 1, it is stated: "our endogenously tagged inx-8(qy78) and inx-9(qy79) strains had dramatically different degrees of embryonic lethality, with qy79 producing over 150 unhatched eggs per worm." When I look at the table, the brood size for inx-9 is ~226 with 41% embryonic lethality. This would lead to on average ~85 unhatched eggs/worm not 150. Where do these numbers come from and which is correct?

We constructed our table in as similar a fashion to Tolkin et al., which reports live brood only in the brood size column as a mean and standard deviation of total live offspring, and report embryonic lethality as a percentage of all offspring (live worms+unhatched eggs), though the total numbers of unhatched eggs is never presented. We have now presented our findings differently in the revised version of the manuscript, with total number of unhatched eggs (as well as percent embryonic lethality) listed alongside live brood.

Given that there are differences between the labs in the behaviors of different strains, it is critical that Kacy et al. perform the control brood sizes on the inx-8 and inx-9 described in lines 231 -236 since a critical aspect of their interpretation hinges on this.

Thank you for pointing out the need to clarify this text. The text in line 231-236 cites prior work on a different set of innexin mutants (Starich and Greenstein, 2020 https://doi.org/10.3390/biom10121655), which refers to broods in the mid-200s as “nearly wild-type”. We referred to this qualitative assessment to contextualize the brood sizes of the transgenic strains in our analysis. We agree with the reviewer that introducing discussion of these other inx-8 mutants is distracting. The text now reads in lines 275-277:

“All of the fluorescently marked strains have mildly to moderately reduced brood sizes. On the basis of brood size alone, there is not a strong reason to prefer one of these markers over another.”

Lines 239-240, please provide references for other brood sizes conducted in the lab.

This line referenced unpublished observations in our lab and previously in the Sherwood lab and has been removed. The numbers Tolkin et al. report—a brood size of 108 with Day 1 embryonic lethality of 87.7%--would be impossible to miss during routine culture and would make performing crosses with this allele difficult. We have seen no such brood size defect in our years working with the strain.Tolkin et al. now drop this claim.

What do you mean in Figure 4 by aberrant engulfment? It seems it is just pointing to foci at the cell membranes that could be junctions between the cells.

Thank you for pointing out the need to better describe this phenotype; Reviewer 1 had the same question, which we address above in the figure included in this letter.

Lines 114-121 should refer to an image figure (3C,D).

Call out to figure 3A has been added, thank you!

Reviewer #3:

In this manuscript, Li et al. describe their further investigation of somatic Sh1 cells association with underlying germ cells. This work provides further support for the model where the distal border of Sh2 cell is forming an interface with DTC and Sh1 overlays the mitotic germ cells poised for differentiation. These findings confirm and extend the previous report, but do not test the functional significance of Sh1/germ cell association. Additionally, through the analysis of coexpressing CED-1::GFP and mKate::INX-8, this work revealed that two Sh1 cells may have drastically different levels of CED-1::GFP expression, while maintaining relatively similar mKate::INX-8 levels. The distinct regulation of transgene expression and asymmetric architecture of Sh1 cells around the germ cells have not been appreciated before, but the functional significance of these findings is unclear. The authors argue that overexpression of CED-1::GFP is detrimental as it is associated with gonad morphology defects, and further propose that high levels of CED-1::GFP expression in distal Sh1 are not well-tolerated.

Overall, this manuscript presents findings that confirm the previously published model; however, several discrepancies between this manuscript and Tolkin et al. preprint are concerning:

1) bcIs39 CED-1::GFP transgene was reported to perfectly overlap with mKate::INX-8 in >85% of cases by Tolkin et al., but only in 60% of cases in the current work.

The “>85%” figure from Tolkin et al., Figure 2 Supplement 2B is derived from 11/13 gonads where the two markers overlap. The image they select to illustrate an example of nonoverlapping expression indeed does show overlapping expression, however, they have just underexposed or underscaled the GFP signal in the distal portion of the sheath (see visible Sh1 features). This observation informed our approach, and instead of looking for cases of “non overlapping expression” we counted cases where two vastly different GFP expression intensities in the two Sh1 cells could lead to a similar inadvertent mis-scaling. We report discrepancies in the coexpressed markers in 6/15 cases in a dataset that was collected for this particular experiment (imaging through the full thickness of the gonad with long exposure times to see the entirety of both Sh1 cells). In other DG5131 datasets, we see discrepancies in expression levels between cells in 5/10 gonads (imaged by Singh on 10/8) and 7/16 gonads (imaged by Gordon, 10/5).

2) The reported brood size and embryonic lethality for several identical or equivalent strains are vastly different. Specifically, NK2571 inx-8(qy78) brood size is 220 vs 155 with embryonic lethality of 8% vs 58% and DG505 embryonic lethality is 20% vs 1%. The causes for these differences need to be identified (culture conditions? temperature? formulation of culture plates?); otherwise these problems will persist/emerge for any other research group using these markers leading to issues with result reproducibility. In contrast to the assertion in Line 243, Tolkin et al. have assessed qy78 allele for brood size and embryonic lethality by itself after an outcross or in the NK2571 strain containing cpIs122 transgene, not in combination with bcIs39. Therefore, one cannot argue that brood size and embryonic lethality defects originated from the genetic interaction of qy78 and bcIs39.

Tolkin et al. have now dropped those criticisms. However, because the Tolkin paper used “wild type” to refer to their strain carrying the bcIs39 marker and DTC marker, we were unsure about the precise genotypes they measured brood size for. Unfortunately, we did not have access to the strains that Tolkin et al. refer to as “qy78” in their table, which are described as a 1x and 2x outcross of our NK2571 strain to “wild-type males”. We recapitulated this experiment by outcrossing to wild-type N2 males, and again we see no dramatic embryonic lethality phenotype caused by the qy78 allele (see first section of letter).

Additionally, a detailed analysis of CED-1::GFP marker of Sh1 in Figure 4 revealed that ~50% of morphologically-normal gonads display an interface between DTC and Sh1, while remaining morphologically-normal gonads show a gap between these cells. This provides an opportunity to test the assertion of the model put forth by Gordon et al. 2020 and challenged by Tolkin et al. preprint – that the distal boundary of Sh1 cells impacts germ cell switch from proliferation to differentiation. According to Gordon et al., 2020 model, the proximal displacement of Sh1 in 50% of gonads expressing CED-1::GFP is expected to shift the position of meiotic entry away from the distal end resulting in a larger distal mitotic region in these germlines. By contrast, data in Figure 5 shows a shorter mitotic region in both strains expressing CED-1::GFP, consistent with Tolkin et al's conclusion that Sh1 position does not affect meiotic entry. Therefore, it appears that while the normal position of Sh1 distal boundary is closer to DTC than previously appreciated, its displacement is unlikely to affect the underlying germ cell population.

In the past we had hoped we could use some of these transgene overexpression strains (specifically our lim-7p::gfp::caax transgenic) to perturb the sheath position, however we strongly suspect that these strains have a failure to label the distal Sh1 cell rather than a displacement of the distal Sh1 cell away from the distal end. We have no way to know for sure given these tools. After observing so many transgenic animals with the GFP patterns observed in Figure 2H and Figure 3B’—and especially 3D—I don’t feel confident concluding that the Sh1 cell is not there, based on absence of transgene fluorescence alone. Absence of evidence is not evidence of absence, as they say. We discuss this in the results, lines 153-174.

However, we did displace the sheath from the distal end by applying RNAi against genes that encode key regulators of branched actin dynamics (arx-2 and unc-60, which encode an Arp2/3 subunit and cofilin, respectively), we don’t shift the proliferative zone as recognized by nuclear morphology, but do shift the region in which GLD-1::GFP accumulates in germ cells (Gordon et al., 2020, Figure 7). We know that Sh1 is not necessary for differentiation (Killian and Hubbard, 2005), but that Sh1-ablated animals are temporally delayed for meiotic entry.

We suspect that the shorter mitotic regions in DG5131 CED-1::GFP;mKate::INX-8 (and to a lesser extent in DG5020 CED-1::GFP) animals reflect impaired germ cell proliferation due to Sh1 abnormality. Recent work that is not affiliated with our group (Gopal et al., 2021) finds that loss of syndecan in sdn-1 mutant alleles and caused by sdn-1 RNAi applied across the late larval-young adult transition shortens the mitotic region of the adult germline and impairs germ cell proliferation; the mutants are rescued by sheath specific expression of sdn-1 (neither DTC nor germ cell expression rescues). Gopal et al. go on to demonstrate that the syndecan phenotype is a glp-1-mediated effect, implicating signaling from Sh1 on the Notch-dependent mitotic germline. This link is easily explained by the morphological overlap of Sh1 and the mitotic germline that Gordon et al. (2020) and Li et al. (under review) describe, and it is harder to explain if Sh1 does not contact the mitotic germline in adults, as Tolkin et al. claim.

Other suggested revisions:

1. Line 180: the conclusion that bcIs39 "sensitizes worms for gonad morphology defects" is unwarranted as disruption of DTC migration appears similar in both described genetic backgrounds. It appears that bcIs39 directly disrupts DTC migration.

Thank you for this suggestion; the text (line 219-220) now reads: The lim-7p::ced-1::GFP transgene seems to cause incompletely penetrant gonad morphology defects.

2. Line 196: remove "of".

3. Line 209: using CRISPR/Cas9 *to* introduce… (add "to").

4. Figure 1 and legend: include the allele designations of edited inx-8 and inx-9 for consistency with other figures.

Thank you! Strains and alleles given.

5. Figure 1D: The position of Sh1 distal boundary in the right column (restrictive temperature) is hard to judge; the dashed line indicates a distal projection in the middle that is not apparent by diffuse GFP signal.

We agree that the merged image is a little difficult to read because of the strong germ cell nuclear expression, but the single channel image in the middle should make it clear that the Sh1 GFP expression forms a continuous sleeve over the distal germline. We mark all Sh1 distal edges with a dashed yellow line to help.

6. line 546, 557, 564 and Figure 1E: I don't think "gc transition" is an accepted term in the field. Perhaps replace with "mitotic cell population boundary"?

You’re right, we have updated to call it the “germ cell transition zone”. “Transition zone” is customary, and we clarify that this is a germ cell feature for those less familiar with the system.

7. line 571: the legend indicates strain ID only for DG5020; is this necessary? If so, all strain IDs need to be included.

Thank you, we agree that all strain ID should be given.

8. line 599: change panel to (D).

9. line 609: change panel to (E).

Thank you very much for catching these!

https://doi.org/10.7554/eLife.75497.sa2

Article and author information

Author details

  1. Xin Li

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Data curation, Formal analysis, Investigation, Visualization, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Noor Singh

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Data curation, Formal analysis, Investigation, Visualization, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Camille Miller

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Data curation, Supervision, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  4. India Washington

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Bintou Sosseh

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Kacy Lynn Gordon

    Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    kacy.gordon@unc.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0967-4020

Funding

National Institute of General Medical Sciences (1R35GM147704-01)

  • Kacy Lynn Gordon

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank T Tolkin, A Mohammed, T Starich, Ken CQ Nguyen, David H Hall, T Schedl, JA Hubbard, and D Greenstein for sharing their manuscript and strains DG5020 (combining published alleles bcIs39 and naIs37) and DG5131 (combining published alleles qy78, bcIs39, and naIs37). We thank D Greenstein and the CGC for the temperature-sensitive mutant strains and B Goldstein and A Pani for LP530. We thank R Dowen and P Breen for anonymized brood size replication experiments. We are grateful for helpful conversations with D Sherwood and other colleagues. Funded by NIGMS Grant 1R35GM147704-01 to KLG.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Yukiko M Yamashita, Whitehead Institute/MIT, United States

Reviewers

  1. Yukiko M Yamashita, Whitehead Institute/MIT, United States
  2. Judith Yanowitz, Magee-Womens Research Institute/ U. Pittsburgh School of Medicine, United States
  3. Ekaterina Voronina, University of Montana, United States

Publication history

  1. Preprint posted: November 8, 2021 (view preprint)
  2. Received: November 16, 2021
  3. Accepted: August 8, 2022
  4. Version of Record published: September 12, 2022 (version 1)

Copyright

© 2022, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Xin Li
  2. Noor Singh
  3. Camille Miller
  4. India Washington
  5. Bintou Sosseh
  6. Kacy Lynn Gordon
(2022)
The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone
eLife 11:e75497.
https://doi.org/10.7554/eLife.75497

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