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.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 , 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] [lag-2p::GFP] [The ur group  used two7p::GFP]  or its p2p::GFP] ), Sh1 ceHere, 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 . Finallysygl-1 and lst-1  gonads, a shift to the restrictive temperature causes proliferation to halt and germ cells to remain stationary within the gonad  of germ cells are associated with Sh1. Here, we describe the original findings and their interpretations, and then our new findings. First, an al cells . In a wial cells , left. Ihe gonad , right. 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  alleles to stop Notch signal transduction and observe where and when germ cells acquire features of differentiation (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 (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.The second set of experiments using temperature-sensitive alleles to reveal differences in germ cell fate along the distal-proximal axis uses ntiation . The samntiation  found thnt study  used a sglp-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  and inx-9(qy79[GFP::inx-9]) alleles (ina-1(qy23[ina-1::mNeonGreen]) (cam-1(cp243[cam-1::mNeonGreen]) . The position of this boundary  coincides with the domain in which germ cells leave the stem cell niche  , was brinGreen]) . For botll niche   show a DTC-Sh1 interface, and half show a bare region .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 . Exposurhenomena . It was lim-7p::ced-1::GFP is coexpressed with the mKate-tagged innexin inx-8(qy78) in strain DG5131 . 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 , the intensity of CED-1::GFP is notably low  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 Just because ried out . Brieflyried out  reportedlim-7p::ced-1::GFP transgene. Interestingly, despite being genetically redundant genes (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.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 nt genes  tagged iinx-8(qy78) and DTC marker has a normally patterned distal germline  that is indistinguishable from wild-type N2  allele at the permissive temperature shown in 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 , 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 . The lenWe also counted the number of mitotic figures made by dividing cells in metaphase and anaphase in these strains  and the 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 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 . Therefore, we consider the results presented here to be confirmatory of the foundational finding of glp-1 expression . This preprint initially reported a severe brood size and embryonic lethality defect in strains bearing the qy78 allele  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.This work was inspired by a recent preprint , 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  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.In the rest of Figure 2D-F, Tolkin et al. use a 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 . Perhaps a more generous annotation would reveal more of the thin somatic cell structures that we know from live imaging are there . 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.fasn-1::gfp , acy-4::gfp , and lim-7p::gfp ; we report ina-1::mNG , cam-1::mNG , and lim-7p::GFP::CAAX . The challenge with definitively proving a more proximal boundary of Sh1—after seeing images like those described in our Finally, each study furnishes additional strains with Sh1 fluorescence expression terminating in variously distal or more proximal domains: Tolkin et al. use bcIs39). These ‘Class 2’ gonads have the pattern that we first reported for endogenously tagged sheath proteins (see 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. Taken together, we suspect that we are seeing more or less the same things but describing them differently. eins see , includiC. 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 . A recent study  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 , howeverlim-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.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 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.In strain descriptions, we designate linkage to a promoter with a We focused on young adult animals around the time egg laying commences, as in 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 . Two replicates of this experiment were performed with the results combined in 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 .Worms from the for 6 hr . PermissDAPI staining was done by modifying standard protocols , with thAll 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 . RFPs weFor quantitative comparisons of fluorescence intensity shown in 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 . A DTC-Slim-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 . 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.The variability of the 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 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 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 .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  or Prism 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.This work extends the previous findings by the authors that suggested the lack of 'bare region' in the  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review: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).Thank you for submitting your article "The The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.eLife paper . However, there are important issues brought up by reviewers that need to be addressed as you can see below.The reviewers agreed that the manuscript represents an important advance that is based on the previous 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 , 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  - 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  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 , 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  uses this transgene in all of the backgrounds analyzed , 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  or bent and deformed . 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 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). 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 , 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  - 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 . 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  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  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 concordantqy78 allele on its own without bcIs39(lim-7p::ced-1::gfp) in the background  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  “NA” in . Also, wqy78;cpIs122) has a proliferative zone that is statistically indistinguishable from wild type . 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.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.. 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.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 . 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 typeReviewer #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 , 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):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.”“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 qy78 and other fluorescently marked strains in Table 1 and discussion thereof.We report and discuss the reduced brood sizes of We have also increased our sample sizes .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.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 . These proteins localize to the cell membrane.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 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.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 . 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 , 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 , which Starich et al. 2014 report in their antibody staining experiments . 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 . 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 . 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  uses this transgene in all of the backgrounds analyzed , 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.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)).Thank you for pointing out the need to correct “all” to “most” (line 396). Tolkin et al. do indeed use 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 . The internal inconsistency between these two markers is not discussed in that study.Our results for 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 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 in Gordon et al., 2020  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 , 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.We also analyzed this 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  or bent and deformed . 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.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 . 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 1, we are not visualizing dedicated DTC markers. Fluorescence from the 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  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.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 .The results from the 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.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 , and we see the same result that we obtained with the 18 hour control, as well as a 21 hour control we recently performed . This suggests that by 36 hours post mid-L4, the relationship between the stem cell zone and Sh1 cell are fixed.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.On the other hand, the original 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 . 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?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  listed alongside live brood.We constructed our table in as similar a fashion to Tolkin et al., which reports 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.inx-8 mutants is distracting. The text now reads in lines 275-277: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 , 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 “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 .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 .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.qy78 allele (see first section of letter).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 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.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.In the past we had hoped we could use some of these transgene overexpression strains , 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 . We know that Sh1 is not necessary for differentiation , but that Sh1-ablated animals are temporally delayed for meiotic entry.However, we did displace the sheath from the distal end by applying RNAi against genes that encode key regulators of branched actin dynamics . 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.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  finds that loss of syndecan in 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.lim-7p::ced-1::GFP transgene seems to cause incompletely penetrant gonad morphology defects.Thank you for this suggestion; the text (line 219-220) now reads: The 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!


Glucocorticoids are widely used for a variety of diseases, but the prevention of glucocorticoid-induced osteoporosis is sometimes neglected. Therefore, the effectiveness of a computerized clinical decision support system (CDSS) to improve the performance rate of preventive care for glucocorticoid-induced osteoporosis was evaluated. We conducted a prospective cohort study of outpatients who used glucocorticoids for three months or longer and who met the indication for preventive care based on a guideline. The CDSS recommended bisphosphonate (BP) prescription and bone mineral density (BMD) testing based on the risk of osteoporosis. The observation period was one year (phase 1: October 2017–September 2018) before implementation and the following one year (phase 2: October 2018–September 2019) after implementation of the CDSS. Potential alerts were collected without displaying them during phase 1, and the alerts were displayed during phase 2. We measured BP prescriptions and BMD testing for long-term prescription of glucocorticoids. A total of 938 patients  were included, and the baseline characteristics were similar between the phases. The median age was 71 years, and men accounted for 51%. The primary disease for prescription of glucocorticoids was rheumatic disease (28%), followed by hematologic diseases (18%). The prevalence of patients who needed an alert for BP prescription  and the acceptance rate of BP prescription  were similar between the phases. The number of patients who had orders for BMD testing was significantly increased  after CDSS implementation. The number of patients who needed an alert for BMD testing was significantly decreased from 93% in phase 1 to 87% in phase 2 (P = 0.004). In conclusion, the CDSS significantly increased BMD testing in patients with a higher risk of glucocorticoid-induced osteoporosis, but did not increase BP prescription. However, the implementation rate of CPGs has not been high2 and it has been challenging to provide guideline-based medical care in the primary care field because of the wide range of areas covered3. Compared to specialists who are more likely to implement CPGs because of the focused range of their field, it was challenging for generalists to implement a CPG for patients with multiple comorbid diseases4.Clinical practice guidelines (CPG) have been considered to have a central role in improving the quality and efficiency of healthcare5 CDSSs have been reported to increase prophylaxis of venous thromboembolism in inpatients7 and improve documentation of asthma care in the emergency room8. CDSSs provide assessments or recommendations similar to those from expert physicians based on the CPG9. These changes in practice with CDSS were reported for predetermined preventive care or documentation of risk populations, but the change in management based on the individual treatment status according to a CPG using a CDSS has not been reported.Computerized clinical decision support systems (CDSSs) have been reported to provide evidence-based recommendations based on patient-specific information and enhance clinical performance for drug dosing and preventive care11. CDSSs have been widely utilized for the management of primary osteoporosis12, but not for the management of glucocorticoid-induced osteoporosis. The management of glucocorticoid-induced osteoporosis is more complicated because physicians of many subspecialties are involved, and the CPG is not straightforward. Several interventions other than CDSSs, such as direct feedback or education, were reported, but the effectiveness of CDSSs in the management of glucocorticoid-induced osteoporosis was not reported14.Glucocorticoids are widely used for a variety of diseases, but the prevention of glucocorticoid-induced osteoporosis has been challenging. Although the treatment of glucocorticoid-induced osteoporosis is well established, and a CPG for this is widely recognized, management of glucocorticoid-induced osteoporosis is suboptimalWe thus hypothesized that CDSSs could improve adherence to the CPG for the prevention of glucocorticoid-induced osteoporosis, developed a CDSS for glucocorticoid-induced osteoporosis, and evaluated its effectiveness for improving management based on the rates of BP prescription and BMD testing in a prospective cohort study.We conducted a prospective cohort study at a tertiary care teaching hospital in Japan. It had 618 beds with 28 departments equipped with electronic medical records and a computerized ordering system. The study was conducted from October 1, 2017 to September 30, 2019, and the computerized clinical decision support system (CDSS) with guideline-based glucocorticoid-induced osteoporosis management was implemented on October 1, 2018. The study period was divided into 1 year before implementation (phase 1: October 2017–September 2018) and 1 year after implementation (phase 2: October 2018–September 2019).15. It advised that patients who took a glucocorticoid for more than 3 months should be prescribed a BP based on their risks, and we modified the guideline to develop the inclusion criteria and the CDSS to be easily applicable in daily clinical practice  prescription at any point during the study period were included. The criteria for BP prescription were derived from the guideline for glucocorticoid-induced osteoporosis issued by the Japanese Society for Bone and Mineral ResearchThe CDSS also recommended BMD testing if it had not been done in the past year. The skeletal areas for BMD testing were either spine, proximal femur, or both according to the physician in charge.The index date was the day of glucocorticoid prescription when the duration of glucocorticoid prescription became 3 months or longer. Patients had multiple index dates if they were prescribed glucocorticoids at outpatient clinics more than once during the study period when the glucocorticoids were calculated to be administered for 3 months or longer at each prescription. When the index date was before October 1st, 2018, such patients were included in phase 1, and patients whose index date was October 1st, 2018 or later were included in phase 2. Although some patients were followed beyond October 1st, 2018 and each index day of glucocorticoid prescription was included in phase 1 or phase 2, each patient was included in either phase 1 or phase 2 according to the earliest index day.This study was approved by the institutional review boards at Hyogo College of Medicine and Shimane Prefectural Central Hospital. According to the policy of the Shimane Prefectural Central Hospital for human researches, written informed consent was substituted by the opt-out on the website for obtaining the information in the medical records. We disclosed the details of the study to the patients and publics in the webpage of Shimane Prefectural Central Hospital and informed the patients of their right to refuse enrollment. All study methods were carried out based on the Japanese Ethical Guidelines for Medical and Health Research involving Human Subjects and Declaration of Helsinki.The electronic medical records with computerized ordering system at Shimane Prefectural Central Hospital is an Integrated Intelligent Management System (IIMS), consisting of electronic medical records, order entry, nursing logs, laboratory and imaging results, prescription data, and hospital claims. We developed the CDSS for glucocorticoid-induced osteoporosis. The physician investigators designed the CDSS and the system engineers constructed the additional codes of the CDSS working on the IIMS. The CDSS obtains the information of prescription data for glucocorticoid for the past 3 months and assess the eligibility of bisphosphonate prescription criteria , laboratory data, primary diagnosis for prescription of glucocorticoid, specialty of physician in charge, and the PSL equivalent dose of the prescribed glucocorticoid. We did not collect racial information because the race was not included in the variables in the guideline for glucocorticoid-induced osteoporosisThe outcome measures were the number of alerts and the actual orders for BP prescription and BMD testing. The acceptance rates were calculated by the number of actual orders for BP prescription or BMD testing divided by the number of alerts for BP prescription or BMD testing, respectively. The alerts for BP prescription and BMD testing were hidden alerts that remained in the background and did not appear on the display in phase 1; thus, orders for BP prescription and BMD testing in phase 1 were considered voluntary.We presented the patient-level variables with medians and interquartile range (IQR) for continuous variables and numbers and percentage for categorical variables stratified by phase 1 and phase 2. We compared patient characteristics between phase 1 and phase 2 using the Chi-squared test for categorical variables and the Wilcoxon rank-sum test for continuous variables. The outcome measures were compared between phase 1 and phase 2 with the Chi-squared test at the patient level and the order level. We used the Fisher’s exact test instead of the Chi-squared test for 2 × 2 contingency table comparison if the expected value in any cell was less than 5. The numbers of alerts for BP prescription or BMD testing were compared between phase 1 and phase 2. The BP prescription rate and the BMD testing rate were calculated as order per alert and compared between phase 1 and phase 2. The numbers of orders for BP and BMD testing monthly were also compared before and after implementing the CDSS.In phase 2, the patient-level variables were compared between those with and without BP prescription among those who received alerts, using the Chi-squared test for categorical variables and the Wilcoxon rank-sum test for continuous variables. The same analyses were done between those with and without BMD testing.Adjusted analyses for patient characteristics were not performed, because their characteristics were considered similar between phase 1 and phase 2. All statistical analyses were performed using JMP version 14.2 . All reported p-values were two-tailed, and p-values less than 0.05 were considered significant.This study was approved by the Institutional Review Board at Hyogo College of Medicine (2495) and Shimane Prefectural Central Hospital (R16-064).During the study period, there were a total of 74,757 outpatients , and 938 patients  met the inclusion criteria and were included Fig. . The mosThe prevalence of patients who needed an alert for BP prescription did not change after implementation of the CDSS, from 306 patients (67%) in phase 1 to 304 patients (63%) in phase 2 (P = 0.24). The prevalence of patients who were prescribed BPs was also similar, 48 (16%) in phase 1 and 57 (19%) in phase 2 (P = 0.33) . The acceptance rate of BP prescriptions for patients was similar between phase 1 (5.5%) and phase 2 (6.2%) (P = 0.35)  in phase 1 to 419 patients (87%) in phase 2 (P = 0.004). On the other hand, the prevalence of patients who had BMD testing was significantly increased, from 18 (4.2%) in phase 1 to 101 (24%) in phase 2 (P < 0.001) . The acceptance rate of BMD testing was significantly increased from 14 (0.5%) in phase 1 to 112 (5.3%) in phase 2 (P < 0.001)  and a history of hip or vertebral fracture in the primary care setting21, but no studies have reported whether a CDSS improved patient outcomes such as fractures. It would take thousands of patients and several years to demonstrate the effectiveness of a CDSS to reduce incidences of rare events. Therefore, it was inevitable that the effect of a CDSS on glucocorticoid-induced osteoporosis would have to be evaluated with process measures, such as BMD testing and BP prescription, in the present study. On the other hand, BP prescription has been shown to reduce vertebral or hip fractures in patients with glucocorticoid-induced osteoporosis in major clinical trials22. In other settings, CDSSs were shown to be effective to prevent venous thromboembolism with appropriate prescription of anticoagulants7. Thus, CDSSs may decrease fractures with evidence-based preventive measures when appropriately implemented and accepted in clinical settings. Another report showed that BMD screening combined with BP prescription was highly cost-effective in primary osteoporosis, and such combination of BMD screening and BP prescription may be effective in glucocorticoid-induced osteoporosis23. Further studies to confirm the effectiveness of CDSSs for glucocorticoid-induced osteoporosis should be considered.CDSSs have been reported to improve process measures such as BP prescription rates in primary osteoporosis24. CDSSs have been reported to improve the adherence rate to CPGs in asthma practice9 and the appropriate prescription rate for renal impairment25. Such documentation, test ordering, and adjustment of pre-existing drugs with CDSSs could be relatively easily accepted by healthcare providers. However, starting a new drug according to a CDSS is a high hurdle, because physicians tend to continue current practice if there are no apparent needs to change25. The additional target of CDSSs could be such new prescription orders based on CPGs.Beyond glucocorticoid-induced osteoporosis, CDSSs have the potential to improve the application rate of CPGs26. Therefore, the usage of BMD testing or BP prescription as outcomes in this study should be acceptable. Third, practice outside the study hospital might have been missed, including medications or examinations at other hospitals. However, the study hospital was the local core hospital, and missing such information was less likely. Fourth, we did not incorporate the data on the history of previous fractures in the CDSS because the history of previous fractures was not systematically documented in the medical record in the outpatient setting. The history of previous fractures is an apparent indication for osteoporosis prevention and thus the benefit of CDSS might not be large. The history of previous fracture is also used to calculate Fracture Risk Assessment Tool (FRAX) and FRAX is easily used to estimate the 10-year risk of fracture27. Although we did not consider the FRAX in this study, we should investigate the efficacy of CDSS incorporating the history of previous fracture or FRAX in the future studies. Finally, this study was conducted at a single center, which limits its generalizability. In addition, the implemented CDSS was developed in this study. Although the decision pathways of the CDSS were simple enough that any electronic medical records or ordering systems could deploy it, the findings should be confirmed in other settings worldwide.This study had several limitations. First, this was an observational study to compare practice before and after implementation of a CDSS. The effects of factors other than the CDSS could not be excluded. Especially, factors in patients should not be ignored. Even if the CDSS recommended and physicians accepted the alerts, patients could refuse the recommendation. As a result, the acceptance rate could be lowered by the patients’ refusal. However, this study was conducted at a single center for a relatively short period of 2 years, and the clinical situation did not change over time. The attitude of patients was also less likely to change during the study period. Indeed, the patients’ characteristics were similar between phases. Second, this study did not evaluate clinical outcomes, such as the incidence of hip or vertebral fractures, when using the CDSS. However, the relationship between low BMD and subsequent fractures has been well documentedA CDSS could improve glucocorticoid-induced osteoporosis practice based on the CPG. The performance rate of guided practice was still less than perfect; thus, further investigation should be conducted to improve the performance rate of guided practice, as well as the patients’ outcomes.Supplementary Information.