MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans

MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans | 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 MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans Xin Li , View ORCID Profile Kacy Lynn Gordon doi: https://doi.org/10.1101/2025.02.24.639896 Xin Li 1 Department of Biology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kacy Lynn Gordon 1 Department of Biology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599 2 UNC Lineberger Comprehensive Cancer Center Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kacy Lynn Gordon For correspondence: kacy.gordon{at}unc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Using a recently published scRNA-seq dataset of adult C. elegans hermaphrodites, we identified a previously unknown regulator of the germ line stem cell niche (the distal tip cell, or DTC). The gene mig-21 has the highest “marker score”–yet no known role–in the DTC. Using classical genetics techniques, RNAi knockdown, and live cell imaging, we discovered that mig-21 integrates information from the Wnt and Netrin pathways to guide anteroposterior and dorsoventral DTC migration. Our study demonstrates the utility of scRNA-seq datasets in revealing testable hypotheses about genetic networks that were masked by redundancy in traditional screening methods. Introduction Single-cell RNA sequencing (scRNA-seq) approaches chemically label cDNA made from transcripts from single cells before sequencing and then identify cell types by transcriptome similarity via clustering algorithm ( Zhang et al. 2023 ). scRNA-seq has rocketed to prominence in studies of developmental biology in the past decade ( Svensson et al. 2020 ). Cell atlases constructed by single-cell RNA-sequencing are being generated for model organisms and human organs across a range of genetic and physiological states, enabling for example the discovery of rare cell populations with importance in human disease ( Plasschaert et al. 2018 ). Ideally, scRNA-seq datasets will lead to new biology being explored well beyond their initial publications. Like genome sequences, these datasets could become precious tools not only for further bioinformatic analyses, but for hypothesis generation for functional genetics studies. We used a recently published C. elegans scRNA-seq dataset ( Ghaddar et al., 2023 ) to study a cell of interest: the C. elegans hermaphrodite gonad stem cell niche, called the distal tip cell (DTC). Each of the two gonad arms of the C. elegans hermaphrodite has a single DTC. The DTC is a migratory germline stem cell niche that has long served as a model for cell migration and stem cell niche biology ( Hubbard and Greenstein 2000 ; Gordon 2020 ). Its stereotyped migration during post-embryonic development patterns the correct U-shaped gonadal morphology of each adult gonad arm. In the L2 and early L3 larval stages, the DTCs migrate away from each other along the ventral body wall in an anterior or posterior direction. In the L3 larval stage, each DTC makes a 90-degree turn off the ventral body wall and then another 90-degree turn onto the dorsal body wall to face the midbody. These turns pattern the bend region of the mature gonad. During the L4 larval stage, each DTC migrates along the dorsal body wall and comes to rest at the dorsal midbody ( Hubbard and Greenstein 2000 ). The genetics of DTC migration and cessation have been studied for decades. DTC migration is governed by two major signaling gradients: a ventral-to-dorsal UNC-6/Netrin gradient that primarily governs D/V migration (the first turn) and a posterior-to-anterior EGL-20/Wnt gradient that primarily governs A/P migration (the second turn) ( Wong and Schwarzbauer 2012 ). Proper A/P and D/V guidance of the DTC results from the integration of inputs from both networks, and they demonstrate some degree of redundancy ( Levy-Strumpf & Culotti, 2014 ). During reproductive adulthood, the DTC is stationary, highly elaborated, and continues to signal to the underlying germ stem cells to maintain them in an undifferentiated state. We asked if previously unknown regulators of the DTC could be identified by examining highly expressed genes detected in that cell type by scRNA-seq. We used the web app WormSeq.org ( https://wormseq.org ) for the dataset reported by ( Ghaddar et al., 2023 ) to look for DTC “marker genes”. Surprisingly, the gene with the highest “marker score” is mig-21 , which has been studied in the context of neuronal cell migration but was not known to function in the DTC. mig-21 was initially identified in a screen for genes that affect touch receptor neuron development (Du and Chalfie 2001), many of which proved to be essential for proper anterior-posterior migration of the progenitors of these cells. MIG-21 is a thrombospondin repeat (TSPI)-containing transmembrane protein that interacts with the Netrin receptor UNC-40/DCC to regulate cell polarization and migration of Q neuroblasts during early larval development ( Middelkoop et al. 2012 ). Like the two DTCs, the two Q neuroblasts initially migrate away from one another along the A/P axis ( Sundararajan and Lundquist 2012 ). MIG-21-dependent polarization of QL and QR restricts the threshold response to the EGL-20/Wnt gradient in these cells ( Middelkoop et al. 2012 ). The role of mig-21 in Q neuroblast migration along the same Wnt and Netrin signaling gradients that guide DTC migration makes it a plausible candidate to examine in the DTC. In this study, we established a role for mig-21 in DTC migration, focusing on the Wnt and Netrin signaling pathways that regulate both Q neuroblast and DTC migration. We also examined whether known interactors of mig-21 in Q neuroblasts coregulate DTC migration. Finally, we examined whether a known regulator of DTC migration cessation interacts with mig-21 . This work provides new insights into the complex interplay of signaling pathways that guide DTC migration and highlights the important role of mig-21 , emphasizing the power of scRNA-seq in hypothesis generation for probing gene function in development. Results and Discussion DTC migration is regulated by mig-21 We identified mig-21 as our gene of interest by looking for DTC-expressed genes with a high “marker score” (genes with relatively high expression levels and relatively specific expression) and “specificity score” (determined by Jensen-Shannon distance)( https://cole-trapnell-lab.github.io/monocle3 ) in scRNAseq results ( Ghaddar et al., 2023 ). The gene mig-21 is at the top of the “marker” list and is third for “specificity” in the DTC ( Figure 1A ). It is the only gene that appears in the top ten genes of both sorts. Download figure Open in new tab Figure 1. mig-21 regulates DTC migration but not cell structure (A) Top 10 genes ranked by marker score (left) and specificity (right) from WormSeq.org ( https://wormseq.org ) for the distal tip cell at the young adult stage. mig-21 is highlighted as the only gene on both lists. (B) Micrographs on the left: DIC imaging of C. elegans hermaphrodites at the late larval L4 stage for mig-21(u787) worms. Micrographs on the right: DIC merged with fluorescence imaging of the same stage mig-21(u787) crossed with a strain bearing a transgene that marks the membrane of the DTC, cpIs122[lag-2p::mNeonGreen:: PLC δPH ], and a nuclear marker inserted at the endogenous lag-2 locus lag-2(bmd202[lag-2::P2A::H2B::mT2]) ( Singh et al. 2024 ). Images are Z-projections through the thickness of the gonad required to capture the whole distal gonad. Black dashed lines outline gonads. Anterior left and ventral down. Yellow asterisks mark DTC; yellow carets mark the proximal vulval position. Scale bar: 20 μm. Yellow boxes indicate the positions of insets shown in E and F. (C) Percentage of DTC migration defects across experimental groups. Low-penetrance but significant defects were identified in mig-21(u787) alone and with fluorescence markers. Wild type N2 strain control (n=3/92), mig-21(u787) (n=23/189), p < 0.05; markers control (n=4/104), mig-21(u787) with markers (n=28/215), p 0.05). (D) Percentage of worms with migration defects in anterior vs. posterior gonad arms of mig-21(u787) worms. Migration defects were more frequent in the posterior gonad arm (n=18/189) compared to the anterior arm (n=7/189), p < 0.05. Anterior-posterior polarity (A/P) defects (n=19/189) were significantly more common than dorsal-ventral polarity (D/V) defects (n=1/189), p < 0.0001. (C-D) All sample sizes refer to individual worms. Error bars represent the standard error of the sample proportion. Statistical analysis was performed using a pairwise proportion test, with p-values adjusted for multiple comparisons via the Benjamini-Hochberg procedure. Significant differences are indicated between groups where applicable. ****p < 0.0001 ***p < 0.001 **p < 0.01; *p < 0.05; ns=no significant. The corresponding sample sizes and statistics are presented in Table S1. (E-F) Enlargement of distal tip fluorescence images from 1B, showing normal (E) and defective (F) migration. Annotations show measurements quantifying DTC morphology and nuclear localization (graphed in 1G-H). White dashed lines represent measurement parameters: (Bottom panels Left) DTC length, as the linear distance from the distal tip to the proximal boundary of the DTC; (Bottom panels Right) nuclear position, as the distance from the distal tip to the geometric center of the nucleus. Scale bars 20 μm except bottom right panels 10 μm. (G) Box plots overlaid with all datapoints measuring the DTC length. Sample sizes refer to individual gonads. Wild type N2 control n=10, and mig-21(u787) n=8. Statistical significance was calculated by unpaired, two-tailed Student’s t-tests, error bars represent ±SEM. No significant difference was observed, t=0.6080, df=16, 95% confidence interval −1.737 to 3.135 μm, p = 0.7374. (H) Box plots overlaid with all datapoints measuring the DTC nuclei position. Sample sizes refer to individual gonads. Wild type N2 control n=10 and mig-21(u787) n=8. Statistical significance was calculated by unpaired, two-tailed Student’s t-tests, error bars represent ±SEM. No significant difference was observed, t=0.4361, df=16, 95% confidence interval −0.2280 to 0.3461 μm, p = 0.7531. First, we examined the effect of mig-21 loss-of-function on DTC migration by analyzing a strain bearing the putative null mig-21(u787) mutant allele ( Middelkoop et al. 2012 ) which has a premature amber stop codon in the extracellular domain. Amber stop codons are not read-through in C. elegans (Patel et al. 2008). In mig-21(u787) mutants, we observed a low penetrance defect in DTC migration in which some DTCs failed to execute the two turns correctly ( Figure 1B-C ). We categorized defects into anterior/posterior (A/P) vs. dorsal/ventral (D/V) polarity defects according to the specific phase of migration that was affected, as well as “other” migration defects and “severe” defects in which gonad growth failed or formed a disorganized mass ( Fig. 1B and Fig. S1A). mig-21(u787) mutants exhibited a significantly higher proportion of A/P defects than D/V defects, with more defects observed in the posterior gonad arm than in the anterior ( Figure 1D ). DTC migration defects are caused by signaling defects ( Levy-Strumpf and Culotti 2014 ), as well as by cell structural abnormalities including those that arise from defects in Rac GTPase signaling ( Singh et al., 2024 ; Reddien & Horvitz, 2000 ) or actomyosin-based contractility and nuclear mispositioning ( Agarwal et al. 2024 ). To further investigate the potential role of mig-21 in influencing DTC shape or nuclear position during migration, we crossed mig-21 mutants with a DTC marker strain that has membrane-localized DTC fluorescence and a nuclear histone tag (lag-2p::mNeonGreen::PLC δPH ; lag-2(bmd202[lag-2::P2A::H2B::mT2]) ( Singh et al. 2024 ). The presence of these transgenes did not enhance DTC migration defects in either a wild-type or mig-21(u787) background ( Figure 1C ). Defects caused by knockdown of Rac GTPase genes were not significantly enhanced by mig-21(u787) (Fig. S1B-D). In the L4 stage, both control and mig-21(u787) mutant DTCs maintained their normal shape and displayed well-defined nuclei polarized in the direction of migration ( Figure 1E-H ), suggesting that mig-21 likely interacts with signaling pathways involved in DTC guidance moreso than cytoskeletal dynamics or nuclear positioning, with a specific impact on anterior-posterior directional polarity, especially in the posterior gonad arm. mig-21 regulates Wnt signaling in the DTC Wnt signaling is the main regulator of DTC and Q neuroblast migration along the A/P axis, the latter dependent on mig-21 ( Sundararajan & Lundquist, 2012 ). Redundancy among Wnt pathway members and between Wnt and Netrin signaling had previously been described by revealing epistasis tests ( Levy-Strumpf & Culotti, 2014 ; Levy-Strumpf et al., 2015 ). Genetic redundancy revealed by superadditive or synergistic interaction does not suggest true molecular redundancy between Wnt and Neterin pathway members, but redundancy in the guidance information they impact. We hypothesized that mig-21 may also function redundantly with Wnt signaling to guide A/P migration, explaining why it had not previously been discovered as a regulator of DTC migration. To assess the functional interaction between mig-21 and the Wnt signaling pathway, RNAi by feeding was used to knock down Wnt pathway genes in the mig-21(u787) background. We targeted mom-5 (which encodes the main Frizzled receptor that acts during DTC migration to guide A/P polarity) ( Levy-Strumpf et al., 2015 ), lin-17 (another Frizzled) ( Levy-Strumpf & Culotti, 2014 ), and five Wnt ligand genes, egl-20 (which forms a Wnt gradient crucial for Q neuroblast migration ( Pani & Goldstein, 2018 ), lin-44 , mom-2, cwn-1, and cwn-2 . We predicted that if mig-21 acts redundantly to the Wnt signaling pathway, we will see an enhancement of Wnt knockdown phenotypes in mig-21(u787) mutants. After empty-vector control RNAi treatment, mig-21(u787) mutants showed the same minor ∼10% A/P migration defect rate we observed on standard growth media. Knockdown of mom-5 /Frizzled with RNAi in wild-type worms caused a ∼20% total per worm A/P migration defect ( Fig. 2A-B ), which agrees with previous mom-5 RNAi results ( Levy-Strumpf et al., 2015 ; Singh et al., 2024 ). In the mig-21(u787) background, the incidence of A/P migration defects after mom-5 RNAi increases to over 50% ( Fig. 2A-B ), with an almost complete bias for the posterior DTC ( Fig. 2C ). Incidence of A/P migration defects caused by lin-17 RNAi treatment of wild-type worms was more modest (<10%), but was also enhanced over twofold by mig-21(u787) with the opposite bias, for the anterior DTC ( Fig. 2C ). We thus conclude that mig-21 functions redundantly with Wnt receptors during DTC migration, playing a bigger role as a counterpart to mom-5 in the posterior DTC and a minor role with lin-17 in the anterior DTC. Because the mig-21(u787) DTC migration defect is so modest, the strong synergistic effect of the double loss of function leads us to conclude that mig-21 and Frizzled genes function redundantly at the genetic level. At the pathway level, we do not propose that MIG-21 may function truly redundantly as a Wnt receptor, but potentially that redundancy among Wnt pathway members themselves depends on mig-21 function. Download figure Open in new tab Figure 2. mig-21 regulates Wnt signaling in the DTC (A) Micrographs: DIC imaging of C. elegans hermaphrodites at the late larval L4 stage, comparing wild type N2 (left) and mig-21(u787) (right) under RNAi control L4440 empty vector, mom-5 RNAi, lin-17 RNAi, or egl-20 RNAi feeding treatment to assess DTC migration defect phenotypes. Images are Z-projections through 2-3 μm showing the distal gonad. Anterior left and ventral down. Black dashed lines outline gonads. Yellow asterisks mark DTC; yellow carets mark the proximal vulval position. Scale bar: 20 μm. (B) All DTC migration defects across experimental groups, including wild type N2 (gray) and mig-21(u787) (black) strains, under RNAi control L4440 empty vector, Frizzled receptors mom-5 and lin-17 and Wnt ligands egl-20, lin-44, mom-2, cwn-1, cwn-2 RNAi feeding treatments. Significant enhancements of migration defects by mig-21(u787) were observed in mom-5 , lin-17 , egl-20 , and mom-2 groups. (C) DTC migration defects for the groups with significant enhancement by mig-21(u787) in anterior (left) and posterior (right) arms. Lighter one means wild type groups, darker one means mig-21(u787) groups. (B-C) All sample sizes refer to individual worms. No defect means no defect observed in that group. Error bars represent the standard error of the sample proportion. Statistical analysis was performed using a pairwise proportion test, with p-values adjusted for multiple comparisons via the Benjamini-Hochberg procedure. Significant differences are indicated between groups where applicable. ****p < 0.0001 ***p < 0.001 **p < 0.01; *p < 0.05; no mark means the comparison was not statistically significant. The corresponding sample sizes and statistics are presented in Table S2. A Wnt ligand-independent function of lin-17 /Frizzled has recently been discovered in the earliest asymmetries in the somatic gonad ( So et al. 2024 ), however, DTC migration is Wnt-dependent ( Levy-Strumpf & Culotti, 2014 ). We next tested genetic interactions of Wnt ligand genes with mig-21 . When they have been investigated previously, a single loss of function of the five Wnt ligand genes causes negligible DTC migration defects ( Levy-Strumpf & Culotti, 2014 ). Indeed, we did not observe major A/P migration defects after wild-type worms were exposed to RNAi for any of the Wnt ligand genes. The mig-21(u787) mutation significantly enhanced migration defects of egl-20 /Wnt and mom-2 /Wnt RNAi. We conclude that mig-21 works partially redundantly with liganded Frizzled receptors (primarily MOM-5) to guide A/P polarity of (primarily posterior) DTC migration, with special sensitivity for EGL-20, the Wnt ligand to which mig-21 regulates the response in the Q neuroblasts ( Middelkoop et al. 2012 ). mig-21 interactors in the Q neuroblasts also act in the DTC In the Q neuroblasts, MIG-21 partners with PTP-3, which encodes a LAR receptor phosphotyrosine-phosphatase (RPTP) ( Harrington et al. 2002 ), to regulate Wnt-responsive cell polarization. ptp-3 is not known to function in the DTC, though its transcripts are detectable in the adult ( https://wormseq.org ) and L2 DTC ( Cao et al. 2017 ). RNAi knockdown of ptp-3 in wild-type worms caused minor incidence of gonad migration defects (<10%, Fig. 3A-B ). However, mig-21(u787) mutants treated with ptp-3 RNAi had a gonad defect rate of ∼20%, with the majority of defects being A/P migration defects in the posterior gonad ( Fig. 3C ). Download figure Open in new tab Figure 3. mig-21 works with ptp-3 and regulates Netrin signaling in the DTC (A) Micrographs: DIC imaging of C. elegans hermaphrodites at the late larval L4 stage, comparing wild type N2 (left) and mig-21(u787) (right) under RNAi control L4440 empty vector, LAR receptor ptp-3 , C-mannosyltransferase dpy-19 and Netrin receptors unc-40, unc-5 RNAi feeding treatment. Images are Z-projections through 2-3 μm showing the distal gonad. Anterior left and ventral down. Black dashed lines outline gonads. Yellow asterisks mark DTC; yellow carets mark the proximal vulval position. Scale bar: 20 μm. (B) All DTC migration defects across experimental groups, including wild type N2 (gray) and mig-21(u787) (black) strains, under RNAi control L4440 empty vector. ptp-3 RNAi, dpy-19 RNAi, unc-40 RNAi, or unc-5 RNAi feeding treatment. Significant enhancements were observed in ptp-3 (p < 0.05) and unc-5 (p 0.05) and unc-40 (p > 0.05) group. (C) DTC migration defects across different experimental groups in anterior (left) and posterior (right) arms (exclude dpy-19 group). Lighter one means wild type groups, darker one means mig-21(u787) groups. Significant enhancement was also observed in posterior arm of ptp-3 (p < 0.01) group. Significant enhancement was observed in anterior (p < 0.01) and posterior (p < 0.05) arms of unc-5 group, with a highly significant increase in the “no turn” phenotype was observed in both the anterior (p < 0.0001) and posterior (p < 0.0001) arms of unc-5 group. While mig-21(u787) does not affect the incidence of gonad defects in the unc-40 RNAi group, it does change the character of migration defects. (B-C) All sample sizes refer to individual worms. No defect means no defect observed in that group. Error bars represent the standard error of the sample proportion. Statistical analysis was performed using a pairwise proportion test, with p-values adjusted for multiple comparisons via the Benjamini-Hochberg procedure. Significant differences are indicated between groups where applicable. ****p < 0.0001 ***p < 0.001 **p < 0.01; *p < 0.05; ns = no significant. The corresponding sample sizes and statistics are presented in Table S3. Several common factors both regulate the DTC and interact with LAR. The guanine nucleotide exchange factor Trio interacts with LAR in mammalian cells ( Debant et al. 1996 ); in C. elegans unc-73 /Trio loss of function phenocopies Rac GTPase loss of function in the DTC ( Singh et al. 2024 ). Substrates of LAR, like Beta-catenin and cadherin, are known as regulators of the post-migratory DTC ( Gordon et al., 2019 ; Tolkin et al., 2024 ). Finally, the LAR receptor mediates adhesion between germline stem cells and the Drosophila male germline stem cell niche ( Srinivasan et al. 2012 ). Future work on the role of mig-21/ptp-3 cooperation in the DTC is warranted. Another interactor of mig-21 in Q neuroblast migration, dpy-19, encodes a C-mannosyltransferase ( Buettner et al. 2013 ). RNAi knockdown of the dpy-19 gene alone or in combination with mig-21(u787) has the same minor DTC migration defect as mig-21(u787) alone, suggesting that any role of dpy-19 in the DTC is likely limited to its processing of mig-21 ( Fig. 3A-B ). Finally, the Netrin receptor UNC-40/DCC interacts with MIG-21/PTP-3 negatively in QR and acts in parallel in QL. We find that wild-type worms on unc-40 RNAi have a ∼30% overall DTC migration defect, including ventralization of migration ( Fig. 3A-B ). When mig-21(u787) mutants are put on unc-40 RNAi, the overall migration defect penetrance does not change or differ between anterior and posterior DTCs, but the nature of the phenotypes changes to include failure to turn at all, and other A/P polarity defects in both the anterior and posterior DTC which are never observed after unc-40 knockdown of wild-type worms, along with notable suppression of D/V polarity defects in the posterior DTC ( Fig. 3C ). Our results suggest that mig-21 and unc-40 have complex and potentially differing interactions in the two DTCs, as they do in the two Q neuroblasts. However, UNC-40 is not the only Netrin receptor in the DTC. mig-21 regulates the Netrin pathway in the DTC The Netrin signaling ligand UNC-6 establishes a concentration gradient along the dorsal-ventral (D/V) axis, directing cellular and growth cone migration ( Norris & Lundquist, 2011 ). This guidance function is achieved through interaction with its receptors, UNC-40/DCC ( Chan et al. 1996 ), and the Netrin receptor UNC-5 ( Leung-Hagesteijn et al. 1992 ). Netrin signaling confers the dominant D/V polarity information in DTC migration, and UNC-5 regulates D/V DTC migration both independently and redundantly with UNC-40 in transducing a repulsive ventral UNC-6/Netrin signal ( Hedgecock et al., 1990 ), and in a Netrin-independent manner ( Levy-Strumpf et al., 2015 ). Since mig-21 alters the nature of unc-40 RNAi defects in the DTC, we hypothesized mig-21 may interact with UNC-5/Netrin receptor during DTC migration as well. Loss of unc-5 /Netrin receptor function causes ventralization of DTC migration in which the DTC migrates out and back along the ventral body, never crossing to the dorsal body wall ( Hedgecock et al., 1990 ). The mig-21(u787) allele alone rarely shows evidence of D/V migration defect (<5%, Fig. 3C ). Treatment of wild-type worms with unc-5 RNAi causes a ∼50% overall ventralization defect ( Fig. 3A ) (with a slight posterior bias, Fig. 3C ), which is in line with previous observations ( Levy-Strumpf & Culotti, 2014 ; Singh et al., 2024 ). Treatment of mig-21(u787) mutants with unc-5 RNAi enhanced per-worm D/V migration defects to over 75% (∼50% each for anterior and posterior DTCs). Notably, over 20% of affected gonads in both the anterior and posterior failed to turn ( Fig. 3C ). The “no turn” phenotype is considered to result from defects of both A/P and D/V polarity of the DTC, with the DTC failing to cross to the dorsal side and then failing to turn back towards the midbody ( Levy-Strumpf & Culotti, 2014 ). We thus conclude that mig-21(u787) enhances the frequency and severity of migration defects caused by the loss of function of UNC-5/Netrin receptor and likely acts redundantly in the same pathway, especially in its contribution to A/P polarity. mig-21 mediates Wnt-Netrin pathway crosstalk Netrin pathway signaling confers the dominant D/V polarity information, and Wnt pathway signaling confers the dominant A/P polarity information during DTC migration, however, the two signaling networks function somewhat redundantly at the genetic level ( Levy-Strumpf & Culotti, 2014 ). Wnt and Netrin pathway loss of function can both mutually enhance and also suppress DTC migration defects caused by loss of function in the other pathway, revealing that each pathway contributes to both proper A/P and D/V DTC migration. Because of its apparent redundancy with both Wnt and Netrin receptor genes, we next wondered if mig-21 might act as a relay or convergence point between the two signaling pathways. We tested how the mig-21(u787) allele affected DTC migration in genetic contexts with combined Wnt and Netrin loss of function. We first generated an mig-21(u787); unc-5(e152) double mutant. The unc-5(e152) allele has a premature stop codon truncating the intracellular domain of all isoforms ( Mahadik & Lundquist, 2023 ; Killeen et al., 2002 ; Hedgecock et al., 1990 ); that intracellular domain mediates the repulsion that brings about the first turn ( Lee et al. 2005 ). The unc-5(e152) mutant and the double mutant have no appreciable A/P migration defect beyond that of mig-21(u787) alone ( Fig. 4A-B ). The unc-5(e152) mutant has an overall D/V migration defect of ∼70% (with a posterior bias, Fig. S2). This is a more penetrant defect than unc-5 RNAi produces, and it is not enhanced by our unc-5 RNAi ( Fig. 4C ), suggesting that the unc-5(e152) mutant is a complete loss of function for unc-5 -mediated regulation of DTC migration. We find that this migration defect rate is not significantly enhanced by mig-21(u787) ( Fig. 4B-C ), providing further evidence that unc-5 (via its C-terminal region) and mig-21 act in the same DTC migration pathway and in the same direction. Download figure Open in new tab Figure 4. mig-21 mediates Wnt-Netrin pathway crosstalk in the DTC (A) Micrographs: DIC imaging of C. elegans hermaphrodites at the late larval L4 stage, comparing single mutants unc-5(e152) (left) and double mig-21(u787); unc-5(e152) (right) mutants under RNAi control L4440 empty vector and mom-5 RNAi feeding treatment to assess DTC migration defect phenotypes. Images are Z-projections through 2-3 μm showing the distal gonad. Anterior left and ventral down. Black dashed lines outline gonads. Yellow asterisks mark DTC; yellow carets mark the vulval position. Scale bar: 20 μm. (B) Comparing the percentage of all classes of migration defects observed across different experimental groups in posterior gonad arms only. (C) All DTC migration defects across experimental groups comparing unc-5(e152) (salmon) and mig-21(u787);unc-5(e152) (cayenne) strains, under control RNAi L4440 empty vector and unc-5 RNAi feeding treatments. (D) Comparing the percentage of only the classes containing anterior-posterior migration defects in posterior gonad arms shown in 4B. Anterior-posterior migration defects include “A/P” polarity reverse and “no turn” categories. Isolating these defects from the “D/V” and “other” classes makes it easier to see the significant suppression of mom-5- RNAi-induced A/P migration defects which is lost in mig-21(u787);unc-5(e152) double mutants on mom-5 RNAi. (E) Comparing the percentage of all the DTC migration defects observed across different experimental groups under lin-44 RNAi feeding treatment. The rate of unc-5(e152) migration defects (n=44/52) was significantly suppressed by lin-44 RNAi (n=34/54). However, this suppression is lost in the mig-21(u787); unc-5(e152) genetic background. (F) Comparing the percentage of the DTC migration defect rates observed across different experimental groups in anterior (left) and posterior (right) arms for samples shown in 4E, with more specific defect categories and classifications. Suppression of unc-5(e152) D/V defects was not observed in anterior arms but was evident in posterior arms. Lighter one means under RNAi control L4440 empty vector, darker one means under lin-44 RNAi. (B-F) All sample sizes refer to individual worms. On the graphs, “no defect” means no defect observed in that group. Error bars represent the standard error of the sample proportion. Statistical analysis was performed using a pairwise proportion test, with p-values adjusted for multiple comparisons via the Benjamini-Hochberg procedure. Significant differences are indicated between groups where applicable. ****p < 0.0001 ***p < 0.001 **p < 0.01; *p < 0.05; no mark means the comparison was not statistically significant. The corresponding sample sizes and statistics are presented in Table S4. With this double mutant, we interrogated the genetic interaction of mom-5 /Frizzled and the unc-5 /Netrin receptor. It is known that mom-5 genetically represses unc-5 (via positive regulation of Rac pathway components ( Levy-Strumpf et al., 2015 )). One key piece of evidence for this conclusion is that mom-5 A/P migration defects are suppressed by unc-5 loss of function ( Levy-Strumpf et al., 2015 ), indicating that mom-5 and unc-5 affect A/P migration in opposite directions, and that unc-5 is downstream of mom-5 in the network. If mig-21 functions primarily as a member of the Netrin pathway, we would expect mig-21(u787) either to slightly enhance the A/P defect suppression or show no effect in a mom-5; unc-5 dual loss of function experiment. On the other hand, if mig-21 primarily acts redundantly with Wnt pathway signaling, we might expect the overall penetrance of A/P polarity defects to increase but for these defects to still be suppressed by unc-5 loss of function. Instead, we see something more complicated. Otherwise, wild-type worms on mom-5 RNAi have a ∼20-30% A/P migration defect ( Fig. 2C and Fig. 4D ). The unc-5(e152) mutant on mom-5 RNAi shows suppression of A/P migration defects to <15% ( Fig. 4B ), as expected. However, the mig-21(u787); unc-5(e152) double mutant on mom-5 RNAi does not suppress the A/P migration defect relative to the mig-21(u787) mutant on mom-5 RNAi alone ( Fig. 4D ). Indeed, mig-21(u787) appears to synergize with unc-5(e152) to convert A/P migration defects to the “no turn” phenotype, which is considered to be a combination of a failed first turn and mispolarized second turn. We conclude that suppression of mom-5 RNAi A/P migration defects by loss of unc-5 is mig-21 -dependent. When we examine D/V migration defects in the same treatment groups, we see that unc-5 loss of function causes substantial ventralization of migration, as expected, and that a substantial fraction of these is enhanced to “no turn” phenotypes by mig-21(u787) , and to a lesser degree by mom-5 RNAi ( Fig. 4B ). Loss of function caused by mig-21(u787) simultaneously enhances A/P and D/V polarity defects caused by loss of function of mom-5 and unc-5 function, respectively. We next tested a treatment by which loss of function of a Wnt signaling pathway member could suppress D/V migration defects caused by loss of unc-5. It had previously been shown that lin-17 /Frizzled and lin-44 /Wnt interact with unc-5 in DTC migration ( Levy-Strumpf et al., 2015 ). That study focused on their redundant regulation of A/P polarity, but shows data that suggest that loss of function of those Wnt pathway members suppresses the D/V defects caused by unc-5 loss of function. When we put the unc-5(e152) mutant on lin-44 /Wnt RNAi, the unc-5(e152) D/V defect rate is suppressed from over 80% to just over 60%. This suppression is completely eliminated in a mig-21(u787) mutant background, with the D/V migration defect identical to the D/V defect rate in the mig-21(u787); unc-5(e152) mutant on RNAi vector control ( Fig. 4E-F ). We thus conclude that suppression of unc-5(e152) D/V migration defects by loss of function of a Wnt ligand is mig-21 dependent. Taken together, these results lead us to conclude that mig-21 plays a key role in balancing the effects of Wnt and Netrin pathway signaling on DTC migration. mig-21 is not required for cessation of DTC migration but sensitizes adult DTCs to polarizing signals We first began investigating mig-21 because RNA-seq data shows that it is strongly and specifically expressed in adult DTCs, however, we went on to identify larval roles for mig-21 in the DTC. We next asked if it interacts with a regulator of the adult DTC, vab-3 /Pax6. This transcription factor is required for the cessation of DTC migration via the transcriptional switch in the alpha integrin subtype expressed by the DTC from ina-1 to pat-2 ( Meighan and Schwarzbauer 2007 ). Loss-of-function of vab-3 causes continued DTC migration in adulthood in which the DTC takes on a meandering or curling path ( Meighan and Schwarzbauer 2007 ). Normally, DTC migration ends at the dorsal midbody, and mig-21(u787) mutants also cease migration in this position ( Fig. 5A ). In otherwise wild-type worms with DTC markers treated with vab-3 RNAi, we see a high penetrance of overmigrated and curling gonad tips ( Fig.5A ) (in excess of 90%, with ∼70% showing extra turns at the tip by 52-55 hrs post L1 arrest, Fig. 5B ). RNAi knockdown of vab-3 combined with the mig-21(u787) allele displays a substantial shift from continued DTC migration with extra turns to DTC overmigration along the dorsal body wall along a straight path ( Fig. 5C-D ). Cessation still fails, but the DTC path stays straighter. If extra turns observed after vab-3 RNAi treatment result from chaotic DTC polarization in response to signaling gradients in the adult, we interpret the suppression of those turns by mig-21(u787) to reflect a role for mig-21 in the continued sensitization of the adult DTC to Wnt and Netrin gradients, just as it is important for sensing and integrating this positional information in the larvae. Download figure Open in new tab Figure 5. mig-21 sensitizes adult DTCs to polarizing signals (C) Micrographs: Confocal fluorescence imaging of C. elegans young adult hermaphrodites expressing ( lag-2p::mNG::PH; lag-2(bmd202[lag-2::P2A::H2B::mT2] ) without (middle) and with (top and bottom) mig-21(u787) under control (top) and vab-3 RNAi (middle and bottom) feeding treatment to assess DTC migration cessation defect phenotypes. Images are Z-projections through thickness of the gonad required to capture the whole distal gonad. Black dashed lines outline gonads. Yellow carats mark proximal vulval position. Scale bar, 20 μm. (B) Comparing the percentage of two main DTC migrate cessation defect rates observed across different experimental groups under vab-3 RNAi feeding treatment. An “extra turn” to “overmigration” defect shift was observed. A robust “extra turn” defect rate marked control on vab-3 RNAi (n=38/57) decreases with mig-21(u787) on vab-3 RNAi (n=24/65), p < 0.01. However, migration cessation is not rescued; a significant increase in “overmigration” defects is observed between marked control (n=4/57) and mig-21(u787) on vab-3 RNAi (n=17/65), p < 0.05. All sample sizes refer to individual worms. Error bars represent the standard error of the sample proportion. Statistical analysis was performed using a pairwise proportion test, with p-values adjusted for multiple comparisons via the Benjamini-Hochberg procedure. Significant differences are indicated between groups where applicable. ****p < 0.0001 ***p < 0.001 **p < 0.01; *p < 0.05; no mark means the comparison was not statistically significant. (C) Cartoon of MIG-21 and genetic interactors from this study, with annotations after ( Norris et al. 2014 ). UNC-6 and SRC-1 (gray) are known to interact with UNC-5, though we have not yet examined their function in the pathway including mig-21 . Conclusions and future directions The nematode-specific mig-21 gene encodes a thrombospondin repeat-containing protein that genetically interacts with Wnt, Netrin, and LAR RPTP receptors during cell migration in both the Q neuroblast cells and–as we have now discovered–in the DTC. In both contexts, mig-21 loss of function more strongly affects the cell that initially migrates to the posterior (this work and Sundararajan & Lundquist, 2012 ), up the Wnt gradient. The molecular basis of these interactions is not known. Previous work ( Middelkoop et al. 2012 ) notes the thrombospondin domains shared by MIG-21 and UNC-5 could potentially mediate direct interactions between MIG-21 and UNC-40/DCC (as UNC-5 and UNC-40 were known to interact ( Lim and Wadsworth 2002 ); the genetic evidence, in that case, supports parallel activity of the receptors ( Middelkoop et al., 2012 ; Sundararajan & Lundquist, 2012 ). In the case of the DTC, our results support mig-21 acting in the same pathway as unc-5, with considerable redundancy in governing ventral repulsion and A/P polarity. We note that the deletion of the C-terminal intracellular domain of UNC-5 is sufficient to abrogate UNC-5-dependent regulation of the first turn (and is known to act via src-1 ( Lee et al. 2005 )), and this deficiency is not compensated for by the presence of MIG-21 (which has an intracellular domain of only 64 amino acids). Deciphering the molecular basis for UNC-5/MIG-21 redundancy in the future should therefore focus on the extracellular domains of these proteins. MIG-21 and the UNC-40/DCC Netrin receptor are thought to regulate Wnt signal response in the Q neuroblast cells by restricting the direction of cell polarization ( Sundararajan and Lundquist 2012 ). It has subsequently been shown that mig-21 together with dpy-19 regulates UNC-40 subcellular localization to the leading edge of the polarizing Q neuroblast ( Ebbing et al. 2019 ). The proposed model by which MOM-5/Frizzled, the Wnt receptor, restricts UNC-5/Netrin receptor activity in the DTC proposed by ( Levy-Strumpf & Culotti, 2014 ) is strikingly similar–a limitation of the direction of cell polarization. Though this has not been demonstrated molecularly yet in the DTC, in other systems, Frizzled protein itself, like UNC-40 in Q neuroblasts, is known to polarize in the cell membrane ( Strutt 2001 ). Our results are most consistent with a model in which MIG-21 functions in the same pathway as UNC-5, and either in parallel or redundantly with MOM-5/Frizzled in conferring A/P polarity, by which it mediates crosstalk between Wnt and Netrin signaling ( Fig. 5C ). Integrating MIG-21 into existing models of DTC polarization implicates several candidates worth pursuing in the future, like the non-receptor tyrosine kinase protein SRC-1 which is recruited by the intracellular domain of UNC-5 and likely leads to cytoskeletal rearrangement in support of proper polarization ( Lee et al. 2005 ), and the ligand UNC-6/Netrin which signals through UNC-5 and UNC-40 ( Norris et al. 2014 ) ( Fig. 5C ). Further work in this area will lead to the integration of known regulators and novel candidates into a more complete model of DTC migration. Methods Sections of this text are adapted K. Gordon lab publications ( Li et al. 2022 ; Singh et al. 2024 ) as they describe our standard laboratory practices . Target gene selection The target gene mig-21 was selected from the single-cell transcriptional atlas of young adult C. elegans WormSeq.org app ( https://wormseq.org ) designed by Ghaddar et al. (2023) . The app offers various tools for gene expression analysis, including but not limited to identifying specific gene markers and assessing gene expression across cell types. In our study, we used the “top gene markers” function first to view the top 100 gene markers for the distal tip cell. This list was generated by the Monocle3 ( https://cole-trapnell-lab.github.io/monocle3/ ) “find markers” function, which “measures specificity using the Jensen-Shannon distance”. We first sorted the candidates by the highest “marker score” which yields genes that are both relatively specific and highly expressed, then we also sorted them by the “specificity” score as recommended by https://wormseq.org . We found that mig-21 had the highest marker score and ranked third in specificity, and was the only gene present in the top ten of both sorts ( Figure 1A ). Strains Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). In strain descriptions, we designate linkage to a promoter with a p following the gene name and designate promoter fusions and in-frame fusions with a double semicolon (::). Some integrated strains (xxIs designation) may still contain for example the unc-119 (ed4) mutation and/or the unc-119 rescue transgene in their genetic background, but these are not listed in the strain description for the sake of concision, nor are most transgene 3’ UTR sequences. The wild-type strain N2 was used as a control. New strains produced for this study are: KLG041 cpIs122[lag-2p::mNeonGreen:: PLC δPH ] II; mig-21(u787) III; lag-2(bmd202[lag-2::P2A::H2B::mT2] ^lox511I^2xHA) V, and KLG042 mig-21(u787) III; unc-5(e152) IV . Worm rearing C. elegans strains were maintained at 20°C on standard NGM media and fed E. coli OP50 for routine strain maintenance. All animals assessed were hermaphrodites, as males have nonmigratory DTCs. Worm populations were synchronized at L1 arrest for developmental staging by standard egg preps ( Stiernagle 2006 ). Confocal imaging All images were acquired on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a ×63 Plan-Apochromat (1.4 NA) objective and an ORCAFusion GenIII sCMOS camera (Hamamatsu Photonics) controlled by microManager ( Edelstein et al. 2010 ). mNG was excited with a 488 nm laser, and mT2 was excited by a 445 nm laser. Worms were mounted on 4% noble agar pads in 0.01 M sodium azide (VWR (Avantor) Catalog Number 97064-646) for live imaging. RNAi E. coli HT115(DE3) containing the L4440 plasmid, either with or without a dsRNA trigger insert sourced from the Ahringer or Vidal Unique RNAi libraries, or our own clone in the case of unc-5 ( Singh et al. 2024 ), were cultured overnight from single colonies at 37°C with ampicillin (100 μg/mL, VWR (Avantor), Catalog no. 76204-346). Subsequently, dsRNA expression was induced with 1mM IPTG (Apex BioResearch Products, cat # 20-109) for one hour at 37°C, followed by plating 200 μl of the culture and incubating overnight at room temperature on prepared NGM plates with a 1:1 ratio (2.5 μL each) ampicillin and IPTG spread uniformly on the surface with a glass spreader. Worm populations were synchronized by bleaching according to a standard egg prep protocol ( Stiernagle 2006 ), plated on NGM plates seeded with RNAi-expressing bacteria as arrested L1 larvae, and kept on RNAi until the time of imaging. RNAi treatment was conducted at 20°C. Image analysis Images were processed in FIJI (Version: 2.14.1/1.54f) ( Schindelin et al., 2012 ). Detailed descriptions of image analysis for different experiments are provided below. Measurements of DTC length and DTC nuclear location The DTCs were identified in the fluorescence images ( Fig. 1E-F ). The length of the DTC was determined as the distance from the gonad tip to the farthest point of the DTC edge. The location of the DTC nucleus was defined as the distance from the anatomical gonad tip to the center of the DTC nucleus. All measurements were obtained using the FIJI straight line tool. Staging and scoring of DTC migration defects L4 ( Figures 1 - 4 , Supplement) or young adult ( Figure 5 ) animals were scored for DTC migration defects based on gonad morphology. In Figures 1 - 4 , we used the framework of ( Levy-Strumpf & Culotti, 2014 ) to categorize defects of A/P polarity, D/V polarity, and “no turn” defects in which D/V turning fails and A/P polarity is reversed. Some cases of A/P polarity defects result in DTC migration into the pharynx (anterior) or tail (posterior) regions, and others involve extra turns in which migration on the dorsal body wall started in these directions and subsequently reversed back towards the midbody. To these we also add a “severe” category in which gonad outgrowth fails completely or the gonad forms a disorganized mass, and an “other” category, usually cases in which the last phase of gonad migration is not maintained along the dorsal body wall. In Figure 5 , vab-3 RNAi causes failure of migration cessation and perpetual migration, and we separate specimens into classic vab-3 phenotypes in which the DTC makes extra turns and cases of “overmigration” in which the DTC maintains its path along the dorsal body wall but overshoots the midbody. Quantification and statistical analysis Statistical tests, sample sizes, test statistics, and p-values for each analysis are provided in the corresponding figure legends. Statistical analysis and multiple comparison corrections were performed using GraphPad Prism Version 10.4.0 (527) for macOS (GraphPad Software, Boston, MA, USA), and Rstudio version 2024.12.0+467 with the rstatix package ( Kassambara 2023 ). The pairwise proportion test was used to compare proportions between experimental groups, and p-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR) at 0.05. For histograms presented in the Figures, the standard error of the sample proportion was calculated with the SEp ^ = √p ^ (1 − p ^ )/n , where p ^ is the proportion of specimens (worms or gonads) of the total observed (n) with the phenotype; error bars reflecting these standard errors (expressed as percentages) were added to each plot using GraphPad Prism. Unpaired Student’s two-tailed t-test was also performed to compare the means of DTC length and DTC nuclear location measurements, with p-values also calculated with GraphPad Prism. Competing interest statement The authors declare no competing interests. Author Contributions X.L. and K.L.G. conceived the project and designed the experiments. X.L. carried out the experiments. X.L. and K.L.G. analyzed the data and wrote the manuscript. Acknowledgments We would like to thank members of the Gordon Lab, especially Noor Singh and Camille Miller, for their technical help and feedback. We thank Rob Dowen and Peter Breen for sharing RNAi clones. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Funded by NIGMS Grant R35GM147704 to KLG. Footnotes Supplemental material is available for this article. References ↵ Agarwal P , Berger S , Shemesh T , Zaidel-Bar R . 2024 . Active nuclear positioning and actomyosin contractility maintain leader cell integrity during gonadogenesis . Curr Biol 34 : 2373 – 2386 .e5. OpenUrl CrossRef PubMed ↵ Buettner FFR , Ashikov A , Tiemann B , Lehle L , Bakker H . 2013 . C. elegans DPY-19 Is a C -Mannosyltransferase Glycosylating Thrombospondin Repeats . Mol Cell 50 : 295 – 302 . OpenUrl CrossRef PubMed Web of Science ↵ Cao J , Packer JS , Ramani V , Cusanovich DA , Huynh C , Daza R , Qiu X , Lee C , Furlan SN , Steemers FJ , et al. 2017 . Comprehensive single-cell transcriptional profiling of a multicellular organism . Science 357 : 661 – 667 . OpenUrl Abstract / FREE Full Text ↵ Chan SS , Zheng H , Su MW , Wilk R , Killeen MT , Hedgecock EM , Culotti JG . 1996 . UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 netrin cues . Cell 87 : 187 – 195 . OpenUrl CrossRef PubMed Web of Science ↵ Debant A , Serra-Pagès C , Seipel K , O’Brien S , Tang M , Park SH , Streuli M . 1996 . The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains . Proc Natl Acad Sci U S A 93 : 5466 – 5471 . OpenUrl Abstract / FREE Full Text Du H, Chalfie M. 2001 . Genes regulating touch cell development in Caenorhabditis elegans . Genetics 158 : 197 – 207 . OpenUrl Abstract / FREE Full Text ↵ Ebbing A , Middelkoop TC , Betist MC , Bodewes E , Korswagen HC . 2019 . Partially overlapping guidance pathways focus the activity of UNC-40/DCC along the anteroposterior axis of polarizing neuroblasts . Dev Camb Engl 146 : dev180059 . OpenUrl ↵ Edelstein A , Amodaj N , Hoover K , Vale R , Stuurman N . 2010 . Computer Control of Microscopes Using µManager . Curr Protoc Mol Biol 92 : 14 .20.1-14.20.17. OpenUrl CrossRef ↵ Ghaddar A , Armingol E , Huynh C , Gevirtzman L , Lewis NE , Waterston R , O’Rourke EJ . 2023 . Whole-body gene expression atlas of an adult metazoan . Sci Adv 9 : eadg0506 . OpenUrl CrossRef PubMed ↵ Gordon K . 2020 . Recent Advances in the Genetic, Anatomical, and Environmental Regulation of the C. elegans Germ Line Progenitor Zone . J Dev Biol 8 : 14 . OpenUrl CrossRef ↵ Gordon KL , Payne SG , Linden-High LM , Pani AM , Goldstein B , Hubbard EJA , Sherwood DR . 2019 . Ectopic germ cells can induce niche-like enwrapment by neighboring body wall muscle . Curr Biol CB 29 : 823 – 833 .e5. OpenUrl CrossRef PubMed ↵ Harrington RJ , Gutch MJ , Hengartner MO , Tonks NK , Chisholm AD . 2002 . The C. elegans LAR-like receptor tyrosine phosphatase PTP-3 and the VAB-1 Eph receptor tyrosine kinase have partly redundant functions in morphogenesis . Dev Camb Engl 129 : 2141 – 2153 . OpenUrl ↵ Hedgecock EM , Culotti JG , Hall DH . 1990 . The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans . Neuron 4 : 61 – 85 . OpenUrl CrossRef PubMed Web of Science ↵ Hubbard EJA , Greenstein D . 2000 . The Caenorhabditis elegans gonad: A test tube for cell and developmental biology . Dev Dyn 218 : 2 – 22 . OpenUrl CrossRef PubMed Web of Science ↵ Kassambara A. 2023 . rstatix: Pipe-Friendly Framework for Basic Statistical Tests . https://cran.r-project.org/web/packages/rstatix/index.html (Accessed January 16, 2025). ↵ Killeen M , Tong J , Krizus A , Steven R , Scott I , Pawson T , Culotti J . 2002 . UNC-5 Function Requires Phosphorylation of Cytoplasmic Tyrosine 482, but Its UNC-40-Independent Functions also Require a Region between the ZU-5 and Death Domains . Dev Biol 251 : 348 – 366 . OpenUrl CrossRef PubMed Web of Science ↵ Lee J , Li W , Guan K-L . 2005 . SRC-1 Mediates UNC-5 Signaling in Caenorhabditis elegans . Mol Cell Biol 25 : 6485 – 6495 . OpenUrl Abstract / FREE Full Text ↵ Leung-Hagesteijn C , Spence AM , Stern BD , Zhou Y , Su MW , Hedgecock EM , Culotti JG . 1992 . UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans . Cell 71 : 289 – 299 . OpenUrl CrossRef PubMed Web of Science ↵ Levy-Strumpf N , Culotti JG . 2014 . Netrins and Wnts Function Redundantly to Regulate Antero-Posterior and Dorso-Ventral Guidance in C. elegans . PLoS Genet 10 : e1004381 . OpenUrl CrossRef PubMed ↵ Levy-Strumpf N , Krizus M , Zheng H , Brown L , Culotti JG . 2015 . The Wnt Frizzled Receptor MOM-5 Regulates the UNC-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells . PLoS Genet 11 : e1005446 . OpenUrl CrossRef PubMed ↵ Li X , Singh N , Miller C , Washington I , Sosseh B , Gordon KL. 2022 . The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone . eLife . https://elifesciences.org/articles/75497/figures (Accessed January 24, 2025). ↵ Lim Y , Wadsworth WG . 2002 . Identification of Domains of Netrin UNC-6 that Mediate Attractive and Repulsive Guidance and Responses from Cells and Growth Cones . J Neurosci 22 : 7080 – 7087 . OpenUrl Abstract / FREE Full Text ↵ Mahadik SS , Lundquist EA . 2023 . A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans . Front Cell Dev Biol 11 : 1240994 . OpenUrl CrossRef PubMed ↵ Meighan CM , Schwarzbauer JE . 2007 . Control of C. elegans hermaphrodite gonad size and shape by vab-3/Pax6-mediated regulation of integrin receptors . Genes Dev 21 : 1615 – 1620 . OpenUrl Abstract / FREE Full Text ↵ Middelkoop TC , Williams L , Yang P-T , Luchtenberg J , Betist MC , Ji N , van Oudenaarden A , Kenyon C , Korswagen HC. 2012 . The thrombospondin repeat containing protein MIG-21 controls a left–right asymmetric Wnt signaling response in migrating C. elegans neuroblasts . Dev Biol 361 : 338 – 348 . OpenUrl CrossRef PubMed ↵ Norris AD , Lundquist EA . 2011 . UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans . Dev Camb Engl 138 : 4433 – 4442 . OpenUrl ↵ Norris AD , Sundararajan L , Morgan DE , Roberts ZJ , Lundquist EA . 2014 . The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP . Dev Camb Engl 141 : 4395 – 4405 . OpenUrl ↵ Pani AM , Goldstein B. 2018 . Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal eds. R. Nusse and M.E. Bronner . eLife 7 : e38325 . OpenUrl CrossRef PubMed ↵ Plasschaert LW , Žilionis R , Choo-Wing R , Savova V , Knehr J , Roma G , Klein AM , Jaffe AB. 2018 . A single cell atlas of the tracheal epithelium reveals the CFTR-rich pulmonary ionocyte . Nature 560 : 377 – 381 . OpenUrl CrossRef PubMed ↵ Reddien PW , Horvitz HR . 2000 . CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans . Nat Cell Biol 2 : 131 – 136 . OpenUrl CrossRef PubMed Web of Science ↵ Schindelin J , Arganda-Carreras I , Frise E , Kaynig V , Longair M , Pietzsch T , Preibisch S , Rueden C , Saalfeld S , Schmid B , et al. 2012 . Fiji - an Open Source platform for biological image analysis . Nat Methods 9 : doi: 10.1038/nmeth.2019 . OpenUrl CrossRef PubMed Web of Science ↵ Singh N , Zhang P , Li KJ , Gordon KL . 2024 . The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis . Curr Biol 34 : 2387 – 2402 .e5. OpenUrl CrossRef PubMed ↵ So S , Asakawa M , Sawa H. 2024 . Distinct functions of three Wnt proteins control mirror-symmetric organogenesis in the C. elegans gonad ed. C. Desplan . eLife 13 : e103035 . OpenUrl CrossRef PubMed ↵ Srinivasan S , Mahowald AP , Fuller MT . 2012 . The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche . Dev Camb Engl 139 : 1381 – 1390 . OpenUrl ↵ Stiernagle T . 2006 . Maintenance of C. elegans . Wormbook 1 – 11 . ↵ Strutt DI . 2001 . Asymmetric Localization of Frizzled and the Establishment of Cell Polarity in the Drosophila Wing . Mol Cell 7 : 367 – 375 . OpenUrl CrossRef PubMed Web of Science ↵ Sundararajan L , Lundquist EA . 2012 . Transmembrane Proteins UNC-40/DCC, PTP-3/LAR, and MIG-21 Control Anterior–Posterior Neuroblast Migration with Left–Right Functional Asymmetry in Caenorhabditis elegans . Genetics 192 : 1373 – 1388 . OpenUrl Abstract / FREE Full Text ↵ Svensson V , da Veiga Beltrame E , Pachter L. 2020 . A curated database reveals trends in single-cell transcriptomics . Database J Biol Databases Curation 2020 : baaa073 . OpenUrl ↵ Tolkin T , Burnett J , Hubbard EJA . 2024 . A role for organ level dynamics in morphogenesis of the C. elegans hermaphrodite distal tip cell . Dev Camb Engl 151 : dev203019 . OpenUrl ↵ Wong M-C , Schwarzbauer JE . 2012 . Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite . Wiley Interdiscip Rev Dev Biol 1 : 519 – 531 . OpenUrl CrossRef PubMed ↵ Zhang S , Li X , Lin J , Lin Q , Wong K-C . 2023 . Review of single-cell RNA-seq data clustering for cell-type identification and characterization . RNA 29 : 517 – 530 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted February 25, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. 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Share MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans Xin Li , Kacy Lynn Gordon bioRxiv 2025.02.24.639896; doi: https://doi.org/10.1101/2025.02.24.639896 Share This Article: Copy Citation Tools MIG-21 is a novel regulator of Wnt and Netrin signaling in gonad migration identified from published scRNA-seq data and functionally validated in C. elegans Xin Li , Kacy Lynn Gordon bioRxiv 2025.02.24.639896; doi: https://doi.org/10.1101/2025.02.24.639896 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13873) Bioinformatics (41887) Biophysics (21424) Cancer Biology (18566) Cell Biology (25465) Clinical Trials (138) Developmental Biology (13365) Ecology (19871) Epidemiology (2067) Evolutionary Biology (24293) Genetics (15591) Genomics (22478) Immunology (17715) Microbiology (40331) Molecular Biology (17150) Neuroscience (88492) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9817) Zoology (2268)