Non-autonomy of age-related morphological changes in the C. elegans germline stem cell niche

Non-autonomy of age-related morphological changes in the C. elegans germline stem cell niche | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Non-autonomy of age-related morphological changes in the C. elegans germline stem cell niche View ORCID Profile Nilay Gupta , View ORCID Profile Mia Sinks , View ORCID Profile E. Jane Albert Hubbard doi: https://doi.org/10.1101/2025.06.13.658151 Nilay Gupta 1 Department Biology, New York University 2 Department of Cell Biology, NYU Grossman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nilay Gupta Mia Sinks 2 Department of Cell Biology, NYU Grossman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mia Sinks E. Jane Albert Hubbard 2 Department of Cell Biology, NYU Grossman School of Medicine Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for E. Jane Albert Hubbard For correspondence: jane.hubbard{at}nyulangone.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Declines in tissue renewal and repair due to alterations in tissue stem cells is a hallmark of aging. Many stem cell pools are maintained morphologically complex niches. Using the C. elegans hermaphrodite germline stem cell system, we analyzed age-related changes in the morphology of the niche, the distal tip cell (DTC), and identified a molecular mechanism that promotes a subset of these changes. We found decreases in the number and length of long DTC processes with age. We also found that a long-lived daf-2 mutant exhibits a daf-16 -dependent maintenance of long DTC processes. Surprisingly, the tissue requirement for daf-16(+) is non-autonomous, and daf-16(+) in body wall muscle is both necessary and sufficient. In addition, after a delay, pre-formed DTC processes deteriorate upon premature germline differentiation, but not upon cell cycle inhibition. We propose a reciprocal DTC-germline interaction model and speculate how reduced daf-2 activity both delays stem cell exhaustion and maintains DTC processes. These studies establish the C. elegans DTC as a powerful in vivo model for understanding age-related changes in cellular morphology and their consequences in stem cell systems. SUMMARY The C. elegans germline stem cell niche morphology is markedly altered with age and is regulated non-autonomously from the muscle by insulin/IGF-like signaling. Results suggest reciprocal niche-germline regulation. INTRODUCTION Age-related changes in cell morphology can alter cell-cell interactions and signaling ( Lopez-Otin et al., 2023 ). Such changes are particularly important to understand in stem cell systems where niche cells can take on complex morphologies and associate intimately with the stem cells they regulate ( Brunet et al., 2023 ). Age-related changes in the cytoskeleton, extracellular matrix, cell adhesion properties, and other factors influence cell morphology and function ( DiLoreto and Murphy, 2015 ). Here, we investigate age-related morphological changes in a simple stem cell niche in vivo . Like many mammalian stem cell systems ( Brunet et al., 2023 ; Lopez-Otin et al., 2023 ), the C. elegans hermaphrodite germline stem cell pool becomes depleted with age ( Garigan et al., 2002 ; Killian and Hubbard, 2005 ; Kocsisova et al., 2019 ; Luo et al., 2010 ; Qin and Hubbard, 2015 ). The niche in this system is a single cell, the distal tip cell (DTC), that caps the end of the gonad ( Fig. 1 ) and produces membrane-bound DSL family ligands, APX-1 and LAG-2, that activate Notch pathway signaling in juxtaposed germline stem cells ( Austin and Kimble, 1987 ; Henderson et al., 1994 ; Kimble and White, 1981 ; Nadarajan et al., 2009 ). The adult DTC morphology is complex, with variable numbers and lengths of long cytoplasmic processes that extend over and between germline stem and progenitor cells ( Byrd et al., 2014 ; Hall et al., 1999 ; Tolkin et al., 2024 ). DTC processes are not essential for establishment of the stem cell pool that occurs during larval stages, but they contribute to contact-dependent Notch signaling in adults ( Lee et al., 2016 ). Download figure Open in new tab Figure 1. The age-related shift in DTC nuclear position and decline in DTC processes number and length have different levels of dependence on daf-2 . (A) Survival curves for daf-2(+) and daf-2(rf) worms with and without the qIs57 [ lag-2p::GFP ] transgene. N2 is the standard wild type, and age is indicated in days post mid-L4 stage. (B) Representative micrographs of DTCs in live worms of the indicated genotypes at adult Day 1, 6 and 10, post mid-L4. The 20 µm region is between the dotted lines, and the area analyzed for number and length of continuous processes (CPs; see Gupta et al., 2024 ; Tolkin et al., 2024 ) is indicated between the dotted and dashed lines, the latter showing the position of the longest CP. Arrows indicate the DTC nucleus. Open arrowheads indicate non-DTC structures. Scale bars are 20 µm. (C) Quantified age-related DTC features (from top left to bottom right): proportion of DTCs with nuclear displacement ≥5 µm (black bar), position of nuclear displacement (µm) from the distal end, proportion of DTCs with any CPs ≥20 µm (black bar), number of CPs ≥20 µm per DTC, mean and maximum length of CPs ≥20 µm per DTC. N values for proportion plots are the same as for the nuclear displacement superplot (top middle). For all superplots, each dot represents a single DTC (n value shown), colors indicate parallel cohorts distinct from those in other main figures, large circles in color are averages for each cohort, the black dot is the pooled average for all cohorts shown, and black line connects pooled average values, indicating the direction and extent of differences between Day 1 and Day 10. Day 10 n values for mean and maximum CP length are lower since only DTCs with CPs >20 µm were included in these measurements. (D) Number, mean and maximum length of CPs in daf-2(rf) at indicated ages in days post mid-L4. For all panels , qIs57 carries lag-2p::GFP and daf-2(rf) is daf-2(e1370). See methods, Tables S2, S3 for statistics details; comparisons are made between Day 1 and Day 10 within a given genotype; for proportion plots, only p < 0.05 are indicated; otherwise, NS is “not significant”, or p ≥ 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. Here we present an aging time-course analysis of changes in DTC morphology and identify molecular mechanisms that delay or hasten them. Our analysis reveals several striking changes to DTC morphology with age. We confirm an age-related proximal shift in nuclear position that occurs at high penetrance (as seen by others under different life history and age conditions ( Kocsisova et al., 2019 ; Urman et al., 2024 )), and that the number and length of long continuous DTC processes decline with age. We examined these phenotypes for their dependence on the DAF-2 insulin-like signaling (IIS) pathway, which influences lifespan. IIS had a relatively minor effect on the nuclear position, but the effects of age on DTC process number and length were highly IIS-dependent. In the daf-2 mutant, long DTC processes persist with age, and this persistence requires DAF-16/FOXO. Surprisingly, the cellular requirement for daf-16 is neither the DTC itself, nor does it appear to be the intestine where daf-16 is implicated in regulating longevity. Instead, daf-16(+) activity in the body wall muscle is both necessary and sufficient to regulate age-related changes to the length of the DTC processes. We also determined that pre-formed DTC processes deteriorate prematurely by differentiating the underlying germ line, but not by impeding cell cycle. We propose a reciprocal DTC-germline interaction feedback model whereby DTC processes contribute to stem cell maintenance while stem cells stabilize DTC processes, supporting mutual maintenance in young adults (and in daf-2 mutants) and mutual collapse with age. RESULTS Adult DTC morphology is altered with age Age-related changes to hermaphrodite DTC features have been reported previously on a short aging timeline and/or in mated worms (to 5 days of adulthood). These include a proximally displaced nucleus ( Kocsisova et al., 2019 ; Urman et al., 2024 ) and an increase in the size, though not number, of gaps seen using a membrane reporter ( Urman et al., 2024 ). We sought to further characterize changes to DTC morphology in intact live self-fertile worms over a more extensive timeline from Day 1 to Day 10 of adulthood, and to define morphometric parameters to describe and compare age-related changes under different genetic conditions. To analyze DTC morphology over time, we selected three time points starting from the mid-L4 as determined by vulval morphology ( Mok et al., 2015 ): Day 1 (24 hours post mid-L4), when DTC elaboration has peaked in the wild type; Day 6 post mid-L4, when the reproductive phase of wild-type self-fertile hermaphrodites is ending; and Day 10 post mid-L4, when worms are on the verge of the steep population decline consistent with their ∼2-3 week lifespan. To minimize variability due to subtle differences in rearing conditions, for each cohort analyzed, worms were selected as mid-L4 larvae on the same day and harvested for live imaging on Days 1, 6, and 10. We examined several different transgenes expressing fluorescent proteins in the DTC, settling on qIs57 which encodes GFP under the control of the lag-2 promoter ( Siegfried et al., 2004 ). Compared to the other transgenes we examined, it revealed a suitably detailed set of morphological features, did not appreciably dim with age, and allowed tracking of the position of the nucleus (Fig. S1). To ensure that the transgene was not itself modulating lifespan, we performed lifespan analyses in parallel with the canonical wild-type strain N2 and found no significant difference ( Fig. 1A ). However, a greater percentage of worms were censored in strains bearing qIs57 (see Materials and methods). Adult DTCs are morphologically variable; two DTCs from each timepoint are shown in Figure 1B and additional examples are in Figure S2. Using our image analysis pipeline ( Gupta et al., 2024 ), we found several striking changes to DTC morphology with age. Day 1 to Day 10 provides the most informative comparisons ( Fig. 1B , C ; see Fig. S3 for additional timepoints). First, recapitulating and extending the findings of others ( Kocsisova et al., 2019 ; Urman et al., 2024 ), we observed that the DTC nucleus, which normally sits at the distal end of the DTC, shifts proximally with age. The percentage of DTCs with the nucleus ≥5 µm from the distal end went from 13% at Day 1 to 77% at Day 10, and these nuclei averaged 3 µm and 9 µm from the distal end at Day 1 and Day 10, respectively. Second, the number and length of processes extending proximally from the DTC ( Byrd et al., 2014 ; Hall et al., 1999 ; Tolkin et al., 2024 ) provided additional parameters that change with age. LAG-2 fusion proteins can be visualized along DTC processes (Henderson et al., 1992; Crittenden et al., 2006 ; Gordon et al., 2019 ) and germ cell proximity to processes is associated with higher probability of signal-responding germ cells ( Lee et al., 2016 ). Therefore, changes to DTC processes could conceivably influence the underlying germ cell fate. Though additional parameters may be relevant, we focused on long DTC processes that appeared continuous with the cell body in maximum projection micrographs (“continuous processes” (CPs); Gupta et al., 2024 ; Tolkin et al., 2024 ). Our methods do not distinguish between previously-defined subclasses of DTC processes that intercalate with or extend along the germ cells ( Byrd et al., 2014 ), but they reflect only gross morphological features including the proportion of DTCs with processes that extend beyond a 20 µm threshold from the distal end, and the numbers and lengths (mean and maximum) of such processes. At Day 1 post mid-L4, all DTCs had at least one process extending beyond 20 µm. By Day 10, 14% of DTCs had no CPs beyond this threshold. Among those DTCs with one or more CP over the threshold, we observed fewer and shorter CPs over time: the mean number of CPs decreased from 11 to 4 from Day 1 to Day 10, as did their mean and maximum lengths (mean 37 to 30 µm, and maximum 60 to 38 µm) ( Fig. 1C ). Insulin/insulin-like growth factor-1 signaling (IIS) promotes age-related changes in DTC process number and length The IIS pathway is highly conserved and modulates longevity in C. elegans , Drosophila , and mice ( Kenyon, 2010b ; Murphy and Hu, 2013 ; Russell and Kahn, 2007 ). In addition to longevity, IIS regulates various physiological processes in C. elegans , including development, metabolism, and stress resistance ( Murphy and Hu, 2013 ). DAF-2 is the sole insulin/IGF-1 receptor in C. elegans ( Kimura et al., 1997 ), and reducing daf-2 activity (e.g. by the reduction-of-function (rf) daf-2(e1370) allele) extends lifespan ( Kenyon et al., 1993 ). To determine whether a reduction in daf-2 activity would affect age-related changes in DTC morphology, we examined DTCs in daf-2(rf) worms. First, we tested whether the qIs57 marker affected the daf-2(rf) longevity phenotype and found that, if anything, the percentage of worms alive between 40-60 days was elevated, but the maximum lifespan was similar ( Fig. 1A ). We aged cohorts of wild-type and daf-2(rf) worms in parallel and measured the parameters as described above. First, we found that daf-2(rf) had a minimal and variable effect on age-related nuclear position displacement ( Figs 1C , S3A). Relative to the wild type, daf-2(rf) displayed a reduced proportion of DTCs that exhibit nuclear displacement of ≥5 µm at Day 10. However, a large percentage (47%) of daf-2(rf) worms nevertheless displayed the nuclear displacement phenotype (versus 77% in the wild type), and the average degree of displacement in microns from the distal end was similar to the wild type in this cohort. The latter finding is consistent with those of Kocsisova et al. ( Kocsisova et al., 2025 , their Fig. S1). In summary, nuclear displacement occurs at a high penetrance in both wild type and daf-2(rf) with an equal distance of displacement, suggesting that IIS plays a minor role in regulating this aspect of age-related changes to DTC morphology. In contrast to the minor effects on nuclear position, reducing daf-2 activity virtually halted age-related changes in CP number and length from Day 1 to Day 10: all daf-2(rf) worms possessed at least one CP ≥20 µm at Day 10, and there was no decrease in the average number of CPs ≥20 µm, nor their mean or maximum lengths, as was seen in the wild type ( Figs 1C , S4). We therefore focused on the DTC phenotypes related to number and length of CPs. The persistence of DTC process number and length in daf-2(rf) does not correlate temporally with extended lifespan or reproductive span In addition to lifespan extension in daf-2(rf) , the reproductive span of self-fertile hermaphrodites averages several days longer ( Dillin et al., 2002 ), and in mated hermaphrodites, the e1370 allele, in particular, delays reproductive aging ( Kocsisova et al., 2025 ). We wondered whether the rate of age-related changes in the number and length of DTC processes might correlate with reproductive span and/or population lifespan. If correlated with reproductive span, we would expect DTC processes to be shorter after a week in daf-2(rf) . If the rate of decline were associated with average lifespan, we would expect to observe shorter processes by 30 days. We therefore collected additional measurements in daf-2(rf) up to Day 30 ( Fig. 1D ; additional timepoints in Fig. S3B). Referencing our lifespan measurements, Day 30 in daf-2(rf) reflects the percent alive of wild type at Day 15. We found that, remarkably, while the number of CPs declined modestly in this cohort by Day 30 ( Fig. 1D ), it still remained above that of the wild type at Day 10 ( Fig. 1C ). In addition, there was no statistically significant decline in the mean length of CPs ≥20 µm up to 30 days. Although we observed a significant decline in the maximum length, the average is still longer than the wild type at Day 10. We conclude that reducing daf-2 markedly delays age-related changes in the number and length of long DTC processes, and that these changes in DTC morphology do not directly correlate with temporal extension of reproductive span or of population lifespan. Insulin/insulin-like growth factor-1 signaling (IIS) acts via DAF-16/FOXO to promote age-related changes in DTC process length Many phenotypes of daf-2(rf) mutants, including age-related phenotypes such as lifespan, depend on the DAF-16/FOXO transcription factor ( Kenyon, 2010a ; Murphy and Hu, 2013 ).To determine whether the daf-2 -dependent age-related changes in DTC process number and length are also dependent on daf-16 , we examined DTC morphology, as visualized with the qIs57 marker, in double mutant strains bearing a daf-16 null mutation daf-16(mu86) and daf-2(e1370) [hereafter referred to as daf-16(0); daf-2(rf) ]. Although the vast majority of DTCs had one or more CP that exceeded 20 µm, the number of processes and their mean and maximum length decreased similar to the wild type ( Fig. 2 ). Download figure Open in new tab Figure 2. Loss of daf-16 largely reverses the effects of daf-2 on CP number and length. From top to bottom: proportion of DTCs with any CPs ≥20 µm (black bars), number of CPs ≥20 µm per DTC, mean and maximum length of CPs ≥20 µm per DTC in the indicated genotypes for worms imaged on Day 1 and Day 10 post mid-L4. N values for proportion plots correspond to the “Number of CPs” superplot to the right; n values for mean and maximum length plots reflect only those DTCs with CPs ≥ 20 µm. For superplots, dots and circles are as described in Figure 1 ; colors indicate cohorts and replicates are distinct from those presented in other main figures. See methods, Tables S2, S3 for statistics details; NS is “not significant”, * p < 0.05, ** p < 0.01, *** p < 0.001, and blue asterisks indicate a significant increase from Day 1 to Day 10. To assess the loss of daf-16 alone, we measured the same parameters in parallel cohorts with the wild type, daf-2(rf) , and daf-16(0); daf-2(rf) ( Figs 2 , S5). In these trials, a small percentage of CPs of Day 10 DTCs in daf-16(0) worms did not exceed 20 µm, though not as many as the wild type. CP number and length also decreased, as in the wild type. Taken together, we conclude that the maintenance of DTC process number and length over time in daf-2(rf) is highly dependent on daf-16 activity. Non-autonomous daf-16 activity influences age-related decline of DTC process length Prior studies indicated that daf-16 activity in specific tissues can influence distinct phenotypes while also contributing to systemic effects. For example, intestinal daf-16 is associated with enhanced stress resistance and increased longevity ( Libina et al., 2003 ; Zhang et al., 2022 ), and daf-16 in the germ line and muscle promotes expansion of the progenitor pool during germline development ( Michaelson et al., 2010 ). Additionally, daf-16 activity in fos-1a expressing cells in the proximal somatic gonad (PSG) prevents the age-dependent decline in number of germline progenitors ( Qin and Hubbard, 2015 ). Therefore, we sought to determine in which tissues daf-16 most influences the maintenance of DTC processes with age that we observed in daf-2(rf) . For this analysis, we focused on the DTC length parameters (mean and maximum). Our first approach took advantage of available extrachromosomal arrays ( Libina et al., 2003 ; Qin and Hubbard, 2015 ). Each includes a tissue-restricted promoter driving a GFP::DAF-16a translational fusion. We performed the aging time course in the daf-16(0); daf-2(rf) background and compared DTC processes length parameters from control and array-bearing (phenotypically Rol) worms, and non-array bearing (phenotypically non-Rol) progeny from the same array-bearing mothers. If daf-16(+) activity were important in a particular tissue location, we expected that its expression would preserve DTC process length relative to daf-16(0); daf-2(rf) . To assess the approach, we first examined CPs in worms bearing an array that expresses daf-16a from the daf-16 promoter. We observed that these parameters (the proportion of worms with DTC CPs over 20 µm, and the CP length – both mean and maximum) were markedly stable relative to daf-16(0); daf-2(rf) ( Fig. 3 ) and to non-array bearing siblings (Fig. S6). The phenotypic similarity of daf-16(0); daf-2(rf) to non-array bearing progeny from array-bearing mothers also suggested that the array did not confer a maternal effect. Download figure Open in new tab Figure 3. daf-16(+) in body wall muscle is sufficient and necessary for daf-2(rf) suppression of age-related changes in DTC process length. (A) Proportion of DTCs with CPs ≥20 µm, and mean length of CPs ≥20 µm per DTC for indicated genotypes ( daf-16(0); qIs57; daf-2(rf) alone and with extrachromosomal arrays expressing daf-16a(+) ) at Day 1 and Day 10 post mid-L4. N values for mean length plots reflect only those DTCs with CPs ≥ 20 µm. See Fig. S6 for additional timepoints and maximum length. (B) Superplots showing mean length of CPs ≥20 µm in genotypes indicated. L4440 indicates the empty vector control and daf-16 RNAi is the vector carrying daf-16 cDNA sequences. See Fig. S8 for additional timepoints and maximum length. (C) Mean length of CPs ≥20 µm in worms raised on 3mM auxin in genotypes indicated, where daf-16::degron is hq389[daf-16::GFP::degron] and Muscle::TIR1 is emcSi71[myo-3p::TIR-1::mRuby] . See Fig. S10 for images, additional time points, and maximum length. For all panels, qIs57 carries lag-2p::GFP , daf-16(0) is daf-16(mu86), and daf-2(rf) is daf-2(e1370). For all superplots, colors indicate cohorts within panels; replicates shown here are distinct from those in other main figures. See methods, Tables S2, S3 for statistics details; NS is “not significant”, * p < 0.05, ** p < 0.01, *** p < 0.001, and blue asterisks indicate a significant increase. We next tested for DTC-autonomy, expressing daf-16a from the lag-2 promoter ( Michaelson et al., 2010 ). Surprisingly, ∼15% of ∼140 DTCs exhibited an unusual morphology at Days 6 and 10, half displaying uncharacteristically long DTC processes, while others displayed one thick CP containing a displaced nucleus (Fig. S7). None of these features were observed under any other condition. Among the remaining ∼85% of DTCs that exhibited normal morphology, the CP length parameters were similar to daf-16(0); daf-2(rf), even though ∼10% of the DTCs in array-bearing worms at Day 10 had no CPs ≥ 20 µm ( Figs 3 , S6). Thus, apart from the low-penetrance alterations in gross morphology, lag-2 -driven daf-16a(+) had no appreciable effect on the persistence of DTC process length with age. We conclude that non-autonomous daf-16a(+) activity is sufficient to prevent the age-related decline in DTC length when daf-2 is reduced. Intestinal activity of daf-16a does not appear to promote age-related DTC length in daf-2(rf) Both heterologous expression approaches ( Libina et al., 2003 ) and protein degradation approaches ( Zhang et al., 2022 ) indicate that the intestine is the major contributor of daf-16 activity that confers extended lifespan in daf-2(rf) . We speculated that changes in DTC morphology might be a function of overall organismal aging. If so, we would expect that intestinal daf-16(+) in the daf-16(0); daf-2(rf) mutant background would stabilize DTC processes, similar to daf-16(+) driven by its own promoter. Again, using the same transgene expressing daf-16(+) that alters lifespan ( Libina et al., 2003 ), we tested the sufficiency of daf-16a expressed from an intestine-specific promoter. We found that although all worms bore DTCs with CPs ≥ 20 µm, CP lengths still declined significantly with age, comparable to that observed in daf-16(0); daf-2(rf) controls ( Figs 3 , S6). In conclusion, although intestinal daf-16(+) contributes the majority of the longevity phenotype in daf-2(rf) ( Libina et al., 2003 ; Zhang et al., 2022 ), it does not appear to regulate the length of DTC processes with age. Neuronal daf-16a(+) activity mildly influences the age-related changes in DTC length Although the intestine is the major contributor of daf-16 on lifespan, there is neuronal contribution as well ( Uno et al., 2021 ; Zhang et al., 2022 ). We tested an array expressing daf-16a(+) from a pan-neuronal promoter and observed that while all DTCs in the worms bearing the neuronally-expressed daf-16a(+) array contained CPs that crossed the 20 µm threshold, the CPs showed a modest but significant length decline with age. We conclude that neuronal activity of daf-16(+) makes a minor contribution to maintaining the length of DTC processes with age in daf-2(rf) . Proximal somatic gonad (PSG) daf-16a(+) activity does not prevent the age-related loss of DTC process length Previous results indicate that daf-16a expressed in fos-1a- positive cells of the proximal somatic gonad (PSG) in a daf-16(0); daf-2(rf) double mutant delays age-dependent loss of germline progenitors ( Qin and Hubbard, 2015 ), and daf-16 -dependent DOS-3, a non-canonical ligand for the GLP-1/Notch receptor, is a relevant PSG signal ( Zhang et al., 2024 ). We hypothesized the PSG-expressed daf-16 might also contribute to DTC morphology. Although prior experiments used naEx239[ pGC629 (fos1p::gfp::daf-16a) + pRF4 ] in daf-16(m26); daf-2(e1370) ( Qin and Hubbard, 2015 ), for consistency between experiments here, we moved the array into the daf-16(mu86); daf-2(rf) background, and measured CP number and length. In short, daf-16a(+) activity in the PSG did not prevent the age-dependent loss of DTC process length. However, these worms were markedly less healthy than other strains bearing daf-16(+) arrays: only ∼50% of worms survived to Day 4, a defect not observed with other daf-16(+) arrays. Moreover, nearly a third (29%) of worms bearing the array had no DTC process ≥20 µm. Of those that did, the mean and maximum lengths were lower than the control on Day 1 and nevertheless continued to decrease with age ( Figs 3A , S6). Collectively, these findings demonstrate that the expression of daf-16a(+) via fos-1a promoter is insufficient to prevent the age-dependent DTC process decline in a daf-16(0); daf-2(rf) background. This result raises the possibility that while daf-16a(+) in the PSG promotes maintenance of the progenitor pool with age ( Qin and Hubbard, 2015 ; Zhang et al., 2024 ), it does not delay the decline of DTC process length with age. Germline daf-16 exerts a minor effect on DTC process length The germ line is the major focus of activity for daf-16 in modulating expansion of the progenitor zone (PZ) in larval stages ( Michaelson et al., 2010 ), and the development of adult DTC morphology is partially dependent on the germ line ( Tolkin et al., 2024 ). To determine whether germline daf-16 is required for long continuous DTC processes to persist in daf-2(rf) , we used RNAi feeding and the rrf-1 mutant in which RNAi is less effective in the soma but is retained in the germ line ( Kumsta and Hansen, 2012 ; Sijen et al., 2001 ). We note that in these experiments (as in Fig. 2 ) daf-2(rf) displays an increase in mean ( Fig. 3B ) and maximum (Fig. S8) CP length from Day 1 to Day 10, an effect that is not observed with rrf-1 alone (Fig. S8). We speculate that this may be related to a delay in the PZ reaching its maximum size in daf-2(rf) worms (see Discussion). As expected from the foregoing mutant analysis, reducing daf-16 by RNAi eliminated the mean and maximum CP length increase observed in daf-2(rf) from Day 1 to Day 10. In the absence of rrf-1 , however, the mean CP length increased in both the L4440 control and with daf-16(RNAi) , suggesting that the contribution of daf-16 from the soma is more relevant for mean CP length. For maximum length (Fig. S8), a minor effect of daf-16(RNAi) is seen in the absence of rrf-1 . We conclude that germline daf-16 plays a minor to negligible role in regulating DTC processes in daf-2(rf) with age. Body wall muscle-specific daf-16a(+) is sufficient and necessary for the DTC process length reduction with age in daf-2(rf) We measured DTC parameters in daf-16(0); daf-2(rf) worms bearing an array expressing daf-16a(+) in body wall muscle ( myo-3p::GFP::daf-16a ; Libina et al., 2003 ). All the worms carrying the array had CPs ≥20 µm ( Fig. 3A ). Furthermore, CP length was maintained in daf-16(0); daf-2(rf) relative to controls without the array and was similar to worms carrying the daf-16p::daf-16(+) array ( Figs 3A , S6). We also did not observe a significant effect of muscle daf-16(+) on the extension of reproductive span that is seen in daf-2(rf) alone nor an increase in self-fertile brood size (Fig. S9A). Together, these findings indicate that array-borne expression of daf-16a in body wall muscle is sufficient to maintain the length of DTC processes with age in daf-2(rf) . To assess the necessity of body wall muscle DAF-16, we used an auxin-mediated degron approach ( Zhang et al., 2015 ). We generated a strain bearing daf-2(rf), a TIR1::mRub y fusion expressed under the myo-3 promoter ( Sabatella et al., 2021 ) together with degron-tagged DAF-16::GFP ( Zhang et al., 2022 ), and the DTC marker. Following the red and green fluorescent protein tags, we confirmed the tissue-specific expression of TIR1 and auxin-dependent loss of DAF-16 (Fig. S10A). In all of the vehicle controls ( daf-2(rf) and the DAF-16::degron alone), the DTCs in daf-2(rf) strains not only maintained but increased in length over the 10 day interval, while mean length of DTC processes in the strain bearing both myo-3p::TIR1 and the degron-tagged DAF-16 decreased ( Figs 3C , S6). Although DAF-16::GFP was undetectable with auxin exposure, the relatively modest effect could be due to residual undetectable DAF-16. These results suggest that daf-16 in body wall muscle is a major contributor to the persistence of long DTC processes in daf-2(rf) with age. Body wall muscle DAF-16a attenuates the rate of germline progenitor zone decline with age Prior studies implicated muscle-expressed daf-16a activity in a minor but significant role downstream of daf-2 in promoting larval expansion of the germline progenitor pool ( Michaelson et al., 2010 ), whereas it did not significantly influence the endpoint of the progenitor cell number in aged worms ( Qin and Hubbard, 2015 ). Our DTC morphology analysis prompted us to re-examine the possibility of a role for myo-3p::daf-16a(+) in maintaining the PZ pool over an aging time-course in daf-2(rf) . We found that the PZ pool of daf-16(0); daf-2(rf) worms expressing myo-3p::daf-16a(+) started with fewer cells than daf-16(0); daf-2(rf) on Day 1, consistent with the results of Michaelson et al. (2010) . Relative to controls, daf-16(0); daf-2(rf) with myo-3p::daf-16a(+) also does not affect the terminal PZ cell count, consistent with the results of Qin and Hubbard (2015) . However, the presence of the myo-3p::daf-16a(+) array does attenuate the rate of decline of the PZ, similar to daf-2(rf) ( Figs 4A , S9B). This result suggests that the slower PZ loss over time in daf-2(rf) is partially dependent on muscle-produced daf-16(+) . Other tissues likely contribute, since an array bearing daf-16(+) driven from its own promoter gives a stronger effect. By contrast, muscle-produced daf-16(+) prevents loss of DTC processes length over time, similar to what is observed with daf-16(+) driven by its own promoter. Download figure Open in new tab Figure 4. Muscle-expressed daf-16 influences both the rate of progenitor zone (PZ) pool loss and continued germ cell divisions with age. (A) The number of PZ nuclei in (n) gonads in worms of the indicated genotypes at Day 1 and Day 10 post mid-L4. (B) The proportion of PZs in Day 10 adults displaying one or more mitotic figures. See Fig. S9C for mitotic index. See Table S3 for all pairwise comparisons. For all panels, qIs57 carries lag-2p::GFP , daf-16(0) is daf-16(mu86), and daf-2(rf) is daf-2(e1370). For all superplots, colors indicate cohorts within panels. See methods, Tables S2, S3 for statistics details; NS is “not significant”, *** p < 0.001. We further asked whether altering daf-2 and daf-16, and in particular daf-16a(+) from muscle, would influence the proportion of gonads displaying active cell divisions (mitotic figures) on Day 10, a time-point at which we observed no mitotic figures in the wild type. We found significantly more daf-2(rf) worms display mitotic figures at Day 10, while daf-16(0); daf-2(rf) do not, though the daf-16(0); daf-2(rf) proportion is intermediate, suggesting that only part of this phenotype may be daf-16- dependent. Of daf-16(0); daf-2(rf) worms expressing myo-3p::daf-16a(+) , 28% display mitotic figures, similar to worms expressing daf-16p::daf-16a(+) . These results suggest that muscle-expressed daf-16a(+) could play a role in maintaining Day 10 germ cell cycling in self-fertile daf-2(rf) hermaphrodites. Overall, muscle-expressed daf-16(+) both delays age-related changes to the PZ pool and to the DTC, with more subtle effects on the PZ than the DTC, though other tissues also contribute to each independently (see Discussion). Germline undifferentiated fate status, but not cell cycle progression, is correlated with maintenance of DTC processes A striking feature of the aged daf-2(rf) germ line is the relative stability of the PZ pool compared to the wild type ( Figs 4A , S9B). In mated worms, daf-2(rf) also displays a more proximal border of SYGL-1-positive stem cells and a more constant position of meiotic entry relative to the wild type ( Kocsisova et al., 2025 ). We further observed that the proportion of gonads with active divisions in Day 10 is higher in daf-2(rf) and the mitotic index did not decline significantly from Day 1 to Day 10, and that all these phenotypes are partially dependent on daf-16 ( Figs 4A-B , S9B-C). These results prompted us to consider the relationship between DTC processes, stem cell fate, and mitotic progression. Out-growth of the DTC processes in early adulthood is dependent on an underlying substrate of the undifferentiated germ cells ( Byrd et al., 2014 ; Tolkin et al., 2024 ). We therefore wondered whether adult DTC processes, once formed, would be stable in the absence of the underlying PZ in early adulthood. We analyzed the number and length of long DTC processes in worms bearing a temperature sensitive allele of glp-1/ Notch, glp-1(e2141), in which the entire PZ differentiates at the restrictive temperature ( Austin and Kimble, 1987 ). We examined the nuclear morphology of distal germ cells and measured DTC parameters from parallel cohorts of worms that underwent the same temperature-shift regime ( Fig. 5A ). After 24 hours at the restrictive temperature, although virtually all distal germ cells had entered meiotic prophase, the CP number and length parameters were unaffected (Fig. S11A-C). By 48 hours, however, the CP length was reduced relative to the wild type ( Fig. 5A-C ). We conclude that premature differentiation of the PZ pool causes a premature decline in the complexity of the DTC morphology, but with a time delay between differentiation and the decline of DTC processes. Download figure Open in new tab Figure 5. DTC processes decline prematurely with germline stem cell differentiation, but not with restricted cell cycle progression. (A, D) Experimental design and images of DAPI-stained control and glp-1(e2141ts) (A) or glp-4(bn2ts) (D) worms at 48 hours after shift to the restrictive temperature. White dotted lines indicate the proximal border of the PZ. Asterisks indicate the distal end. (B, E) Examples of DTC morphology in worms scored in parallel to those shown in panel (A). Black dashed lines indicate the longest CP. Arrows indicate the DTC nucleus. (A,B,D,E) Scale bars are 20 µm. (C, F) Quantification of mean and maximum lengths of CPs ≥20 µm in control and glp-1(e2141ts) (C) or glp-4(bn2ts) (F) worms at indicated time points. Colors indicate cohorts within panels. See methods, Tables S2, S3 for statistics details; NS is “not significant”, * p < 0.05, *** p < 0.001. Download figure Open in new tab Figure 6. Cartoon representation of a two-component feedback model for niche-stem cell interaction at early and late (“old”) adult stages. Dotted box indicates area of direct DTC-germline interaction that is lost with age. White circles represent undifferentiated germ cells, and grey circles represent differentiated germ cells. See text for details. Because differentiated germ cells no longer divide, and because daf-2(rf) display more mitotic figures than the wild type at a late time point (Day 10; Figs 4B , S9C), and this phenotype is partially dependent on muscle-expressed daf-16(+) , we wondered whether the DTC process maintenance requires active cycling in the underlying germline stem/progenitor cells. To test this possibility, we used a temperature sensitive allele of glp-4, glp-4(bn2), that causes germ cell cycle arrest in pro-metaphase; arrested glp-4 germ cells are resistant to differentiation, even in the absence of glp-1 ( Beanan and Strome, 1992 ). Remarkably, after 48 hours at the restrictive temperature, although glp-4 mutant gonads display altered nuclear morphology, the DTC processes did not collapse ( Figs 5D-F , S12A-C). We further observed that germ cells were capable of recovery after shifting back to the permissive temperature for another 48 hours, and the DTC processes remained intact (Fig S12A-C). Therefore, while loss of stem cells to differentiation eventually leads to changes in DTC morphology that resemble what is seen in older adults, interfering with cell cycle without causing differentiation (as in glp-4 at the restrictive temperature) does not. DISCUSSION Here we define age-related changes in DTC niche morphology and assess their dependence on IIS, a molecular mechanism that affects longevity. We found that an age-related shift in DTC nucleus position is only modestly affected by IIS in self-fertile hermaphrodites, as was previously shown in mated worms ( Kocsisova et al., 2019 ), while the decline in the number and length of long DTC processes is highly dependent on IIS. We identify body wall muscle as a major source of a daf-16(+) -dependent activity that can support both the persistence of long DTC processes in a daf-2(rf) mutant and the underlying germline progenitor pool. Finally, we determine a dependency relationship between the germ cell proliferative state and maintenance of pre-formed DTC processes. A model for reciprocal feedback interactions between the aging germline and niche In a seeming paradox, daf-2(+) expands the larval germline progenitor zone (PZ) but drives loss of the PZ with age, and both effects are dependent on daf-16 . In larvae, the germline cell cycle is slower in worms with reduced daf-2 activity and this is dependent on daf-16(+) in the germ line and body wall muscle ( Michaelson et al., 2010 ; Roy et al., 2016 ). In aged adult worms, a larger PZ pool is maintained with reduced daf-2 activity ( Qin and Hubbard, 2015 ; Fig. 4A ). In this case, one daf-16 -dependent signal, DOS-3, from the proximal somatic gonad likely supports GLP-1/Notch activity in the germ line ( Zhang et al., 2024 ). We found that, in addition, both the PZ pool and long DTC processes are maintained in old daf-2(rf) adults in a daf-16(+) muscle-autonomous manner. The exact daf-16 -dependent mechanism – and whether it acts on the directly on the DTC, the germline, or both – awaits further study. We speculate that such a factor could be a daf-16 -dependent secreted factor – e.g., a mitokine or myokine signaling mechanism, or some other mechanism secondary to the IIS role in muscle function ( Herndon et al., 2002 ; Wang et al., 2019 ). There is precedent for body wall muscle-produced secreted proteins acting on the germ line, including HIM-4/hemicentin ( Vogel and Hedgecock, 2001 ) and SWM-1 ( Chavez et al., 2018 ). Regardless of the exact mechanism, these observations suggest a model for interplay between the control of cell fate and cell cycle rate that may contribute to the dynamics of the germline stem cell system over time with age. Several additional relevant features of the system are noted below. First, stem cell fate and cell cycle rate are independently controlled. The PZ consists of a distal-most SYGL-1(+) germline stem cells (GSCs) that are actively responding to GLP-1/Notch ( Kershner et al., 2013 ), as well as their more proximal SYGL-1(-) progeny that complete a cell division before differentiating ( Fox and Schedl, 2015 ). Reducing glp-1 activity moves the border of the PZ distally and reduces the number of GSCs but, importantly, does not alter their rate of cell cycle progression ( Fox and Schedl, 2015 ; Michaelson et al., 2010 ; Roy et al., 2016 ). By contrast, reducing daf-2 slows the cell cycle (e.g., lowers M-phase and S-phase indices, a rough proxy) in larvae but does not influence the stem versus non-stem cell fate decision governed by GLP-1/Notch ( Michaelson et al., 2010 ; Roy et al., 2016 ). Worms with reduced daf-2 activity therefore reach adulthood with fewer cells PZ cells. This pool declines with age, but at a rate far slower than the wild type (Kocsisova et al., 2022; Qin and Hubbard, 2015 ; Fig. 4A ). Second, as in many mammalian stem cell systems ( Brunet et al., 2023 ), C. elegans GSCs become depleted over time ( Kocsisova et al., 2019 ; Qin and Hubbard, 2015 ), and, importantly, the PZ pool is effectively “used up” with age. This loss is accelerated in the presence of replete sperm that encourage germline flux ( Kocsisova et al., 2019 ; Qin and Hubbard, 2015 ). In addition, with age, the mitotic index slows, even in the presence of replete sperm ( Kocsisova et al., 2019 ). Finally, in early adulthood, the outgrowth of DTC processes partially depends on adhesion to underlying proliferative germ cells ( Tolkin et al., 2024 ). Taking these observations together, we propose a two-component feedback system that supports homeostasis of the progenitor pool in early adulthood, and the loss of which contributes to the collapse of both niche morphology and the stem cell pool with age. In this model, the early adult PZ steady state is maintained by a combination of DTC-to-GSC signaling via GLP-1/Notch to designate the stem cell fate in responding germ cells, as well as DAF-2-dependent inputs that promote continued stability of DTC processes. Over time, however, as cells leave the PZ through differentiation (meiotic entry) and the pool of undifferentiated germ cells becomes depleted, overlying DTC processes are not maintained due to both loss of DAF-16-dependent muscle (and other) effects, as well as PZ depletion. Decreased ligand availability due to shortened DTC processes would then further deplete the stem cell pool and further destabilize the DTC processes. In the daf-2(rf) scenario, we propose that in addition to the effect on daf-16 , the persistence of slow-cycling yet undifferentiated germline stem/progenitor cells may contribute to the stability of DTC processes, perhaps by adhesion mechanisms similar to those that act in the early adult ( Tolkin et al., 2024 ). This stability could then support DTC-to-GSC interactions along the DTC processes that help maintain stem cell fate ( Fig. 5D ). Again, GLP-1/Notch signaling afforded by stable DTC processes may, in turn, support germ cells in an undifferentiated, proliferation-competent (and continued slow-cycling) state. Reducing daf-2 would thereby delay the age-related declines in both components of the reciprocal interaction that are mutually supportive, the progenitor pool and niche morphology. Temporally, the decline of the stem cell pool and PZ precedes that of the DTC processes ( Figs 1 , 4). Similarly, when germ cells are forced to differentiate, a time delay occurs before the DTC declines ( Fig. 5 ). We observed that loss of glp-1 , which causes germ cells in the PZ to differentiate, leads to premature collapse of already-formed DTC processes while loss of glp-4 , which impedes the cell cycle but does not cause differentiation does not cause the collapse of DTC processes. These results implicate the undifferentiated state of germ cells in the germline-to-DTC branch of the feedback to help maintain DTC processes. While this model provides a framework for reciprocal signaling between DTC processes and the germ line, additional influences act on the system. For example, the regulation of GLP-1/Notch signaling with age ( Kocsisova et al., 2019 ) is complex: in addition to DOS-3-mediated signaling from the proximal somatic gonad ( Zhang et al., 2024 ), the shift in the position of the DTC nucleus also alters the patterning of signaling ( Urman et al., 2024 ), and there may be germline-autonomous aspects of reduced GLP-1/Notch signaling since, at least to Day 4, sygl-1 RNA levels do not change ( Urman et al., 2024 ). In mated worms, reduced Notch signaling is implicated in depletion of the stem cell pool and reproductive aging, both of which are also delayed in mated daf-2(rf) worms ( Kocsisova et al., 2025 ). It will be of interest to determine, ideally in individual gonads, to what extent reduced ligand signaling due to changes in DTC morphology with age contribute to the age-related decline in the germline stem cell pool and subsequent reproductive aging. Our model also predicts that the effects of DAF-16 from the proximal somatic gonad that act on the germ line should affect the DTC too, via feedback, since they preserve the PZ. However, we did not observe the predicted stabilization of DTC processes when daf-16(+) was expressed in the proximal somatic gonad in daf-16(0); daf-2(rf) . This result, together with the more marked effect of muscle-expressed daf-16 on the DTC versus the PZ suggest that the germ cell interaction is not the sole arbiter of DTC stability with age. Finally, many aspects of the germ cell cycle are unusual, including a highly abbreviated G1, constitutively high levels of cyclin E/CDK2 throughout the cell cycle, and transcriptional regulation of CDK-2 through inhibition of DPL-1 ( Fox et al., 2011 ; Furuta et al., 2018 ). How such cell cycle features are modified with age and how such modifications influence germ cell propensity to differentiate are unresolved. General implications for aging stem cell systems and regenerative medicine Our results demonstrate that niche morphology is a key aspect of aging stem cell systems. Non-autonomous control of niche morphology, by tissues outside the stem cell system and/or the adjacent stem cells, may be important in other stem cell systems as well. Our work further suggests that deeper knowledge of in vivo interactions, especially those influenced by cellular morphology and cell cycle as well as cell-cell signaling, are required to understand stem cell behavior and tissue regeneration, especially in aging systems. MATERIALS AND METHODS Nematode strains and maintenance The C. elegans strains utilized in this study were raised on standard nematode growth media (NGM) agar and E. coli OP50 at 20°C, unless otherwise specified. All strains were derived from Bristol N2 ( Brenner, 1974 ). All strains, including their complete genotypes and sources, are provided in Table S1. The strains generated in this study were obtained through genetic crosses and homozygosity was confirmed by progeny testing – visually for strains bearing fluorescent markers or by DNA sequence analysis based on information in WormBase ( Davis et al., 2022 ). Strains generated for this study are GC1593, GC1607, GC1616, GC1676, GC1682, GC1683, GC1691, GC1692, GC1696, GC1748, GC1778, GC1817, GC1819, and GC1912, and are available upon request. Aging and lifespan assays Multiple plates of approximately 20 L4 hermaphrodites per plate were prepared, and worms were transferred daily to fresh plates to prevent overcrowding and mixing with their progeny. Worms were collected for imaging or PZ counting at intervals indicated. Controls were run in parallel within each cohort; cohorts share a color in each figure that reports DTC morphometrics or PZ nuclei counts, or mitotic index. Lifespan assays ( Fig. 1A ) started with 100 worms per genotype. For N2 and CB1370, ∼15% were censored due to bagging, desiccating on the petri dish wall, or were otherwise missing. For the two strains that contain qIs57 (JK2869 and GC1607) the rate of censoring for the same reasons was higher, at 40-50%. Imaging With the exception of images of GC1412 in Fig. S1, DTC images were acquired using a Apo 60X oil immersion lens on a Nikon W1 spinning disk confocal microscope and subsequently processed, quantified, and analyzed as described in Gupta et al., 2024 ( Gupta et al., 2024 ). The DTC was visualized using the qIs57 GFP marker, with a 488 nm excitation laser set at ≤10% power. The exposure time was set to 100 ms or 1 frame, the EM Gain Multiplier was set to 50, the conversion gain was set to 1, and the readout mode was set to ‘EM Gain 10 MHz at 16-bit’. The images were acquired as Z-stacks at 0.5 µm step size and saved in .nd2 format. The excitation laser used for DAPI was 405 nm at 20-30% power, and for RFP was 561 nm at 20-30% power range. DAPI staining, progenitor zone (PZ) counts, and mitotic figures and index DAPI staining (using Vectashield from Vector Laboratories™ H-1200-10) was performed as described in Pepper et al., 2003, except that fixation and staining were done in a 9-well glass depression plates (Corning™ 7220-85) to minimize the loss of older worms. Images were acquired using a 60X oil immersion lens on a Nikon W1 spinning disk confocal microscope. The progenitor zone (PZ) is defined as the region before the occurrence of more than one crescent-shaped nucleus within the same transverse section of the gonad ( Crittenden et al., 2006 ; Killian and Hubbard, 2005 ). After visualizing the entire stack, the PZ region of interest was identified, and the nuclei were counted manually using the multi-point tool in ImageJ™. Mitotic figures were defined as obvious metaphase, anaphase, and early telophase figures as visualized in images after whole-mount DAPI staining. Mitotic index is the number of mitotic figures over total number of PZ nuclei. Auxin-mediated degradation For auxin-inducible protein degradation (AID) experiments, 3 mM of Indole-3-acetic acid (IAA, auxin) (Thermo Scientific™ A10556.14) or an equivalent volume of carrier EtOH for controls was added to NGM agar prior to pouring plates. OP50 bacteria were concentrated approximately 2x prior to seeding the auxin-containing plates to compensate for auxin inhibition of bacterial growth ( Zhang et al., 2022 ). Worms were cultured on both the auxin-containing plates and the control plates for multiple generations prior to initiating the aging protocol. Analysis of glp-1(e2141) and glp-4(bn2) Worms ( glp-1 and glp-4 mutant with parallel controls) were collected at the mid-L4 stage and cultured at 15°C for 48 and 72 hours, respectively, to allow for DTC elaboration comparable to that seen in Day 1 adults raised at 20°C. For both genotypes, a group of worms were separated from the cohort and imaged (T0). For glp-1 experiments, the rest were shifted to 25°C and imaged 24 and 48 hours later. For glp-4 experiments, the rest were shifted to 25°C for 48 hours and then shifted back to 15°C for another 48 hours. Cohorts of worms that were grown under the same conditions were split and analyzed live for DTC features or for cell counts after DAPI staining. RNAi bacterial feeding RNAi feeding was carried out as previously described ( Timmons et al., 2001 ) with minor modifications. Single colonies of E. coli strain HT115 carrying either an empty vector (L4440) or a vector containing daf-16 cDNA sequences were obtained from frozen stocks on solid LB plates supplemented with 100 µg/ml ampicillin and 10 µg/ml tetracycline, grown overnight at 37°C and used to inoculate liquid LB cultures supplemented with 100 µg/ml ampicillin. Cultures were grown at 37°C with shaking for 6-8 hours and used to seed NGM plates supplemented with 100 µg/ml ampicillin and 0.5% β-lactose. Seeded RNAi plates were kept at room temperature for 24 hours prior to adding worms. Complete penetrance of the bli-3 Bli phenotype was used as a positive control to confirm the efficacy of RNAi reagents in each experimental replicate. Fresh plates were seeded twice a week over the course of the experiment. L4 hermaphrodites were cultured on either L4440-or daf-16 RNAi-seeded plates for 1 generation prior to starting the aging assays. Statistics Table S2 (.xlsx file) provides properties analyzed, means, SE/SD, and n values for all data presented in main and supplementary figures (tab for each main and corresponding supplemental figure). Table S3 (.xlsx file) lists the genotypes compared, statistical tests, and the exact p values for each main and supplementary data figure on a separate tab. For lifespan assays, the Mantel-Cox log-rank test was used. Fisher’s exact test was used for pair-wise comparisons of proportion plots (DTCs with nuclear displacement ≥5 µm, DTCs with any CPs ≥20 µm, and PZs with mitotic figures at Day 10). For determining significance between nuclear displacement, number, mean length, and maximum length of CPs ≥20 µm, the number of PZ nuclei, or mitotic index: two-tailed t-test for pairwise comparisons (main figures), and two-tailed t-test with Bonferroni correction for multiple comparisons (supplement). When reporting the mean and maximum CP length, we did not include DTCs that with no CPs >20 µm. All sample numbers (n) are also reported within figures. FUNDING NIH R01AG065672 NYSTEM DOH 01-C32560GG-3450000 ACKNOWLEDGEMENTS We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), NYU Langone’s Microscopy Laboratory (RRID: SCR_017934), especially Michael Cammer; this shared resource is partially supported by the Cancer Center Support Grant P30CA016087, Sophia Heimbrock for technical assistance, and Anke Kloock, Theadora Tolkin, Julie Manikas and Caroline Goutte for advice and support. Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R01AG065672 New York State Department of Health, NYSTEM , 01-C32560GG-3450000 Footnotes Edits were made to the text and figures, especially to figure 5 and new figure 6. REFERENCES 1. ↵ Austin , J. and Kimble , J . ( 1987 ). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans . Cell 51 , 589 – 599 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Beanan , M. 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Nat Commun 15 , 4904 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 08, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Non-autonomy of age-related morphological changes in the C. elegans germline stem cell niche Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Non-autonomy of age-related morphological changes in the C. elegans germline stem cell niche Nilay Gupta , Mia Sinks , E. 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