We have previously demonstrated that DTC production and SGP polarity remain normal in any Wnt single or compound mutant strains including quintuple Wnt mutants (lin-44; cwn-1; egl-20 cwn-2; mom-2), suggesting that Wnts may not be required for SGP polarity (Yamamoto et al., 2011). However, when a cwn-2 mutation was combined with that of lin-17/Frizzled, we observed that most animals lacked one or both DTCs (a missing DTC phenotype) as determined by the expression of a DTC marker, mig-24::Venus (Tamai and Nishiwaki, 2007). While lin-17(n3091 with nonsense mutation) single mutants exhibit a weak missing DTC phenotype (Table 1; Sternberg and Horvitz, 1988), lin-17; cwn-2 showed a strong enhancement in regard to the absence of the anterior DTC (Table 1: p<0.0001 by Pearson’s chi-square test). We observed a similar phenotype in lin-17(n671); cwn-2 double mutants, confirming that this genetic interaction is not allele-specific. Although mutations in other Wnt genes (cwn-1 and egl-20) by themselves or in combination did not cause such enhancements, the cwn-1 mutation enhanced the phenotype of lin-17; cwn-2 for both the anterior and posterior DTCs (p<0.0001 by Pearson’s chi-square test), resulting in the majority of lin-17; cwn-1; cwn-2 triple mutants lacking both gonadal arms and being sterile. These results indicate that lin-17/Frizzled and Wnt genes (cwn-1 and cwn-2) regulate DTC production through parallel pathways.

We note that the DTC defects in various compound mutants described above and below were more severe at 15°C compared to 22.5°C for unknown reasons. Since the temperature sensitivities of individual mutations have not been reported and most mutations are nonsense or deletions, we hypothesize that the processes for DTC production are somehow cold-sensitive.

We found that lin-17; egl-20 cwn-2 triple mutants exhibited a unique phenotype that has not been reported before. The triple mutants frequently displayed two DTCs in the posterior region accompanied by the absence of a DTC in the anterior region (this phenotype is hereafter referred to as Dpd for double posterior DTCs) (Figure 2). We have not observed triple mutants with three DTCs. These observations suggest that the Dpd phenotype is caused by the miss-migration of the anterior DTC derived from the Z1 cell, rather than the extra production of posterior DTCs from the Z4 cell. More analyses of the mis-migration phenotype are shown below. In Table 1, we considered that Dpd animals have both anterior (Z1-derived) and posterior (Z4-derived) DTCs.

lin-17; cwn-1; egl-20 cwn-2 quadruple mutants exhibited a similar phenotype to lin-17; cwn-1; cwn-2 triple mutants with most animals lacking both DTCs. However, the Dpd phenotype was not observed in the quadruple mutants.

In contrast to the pronounced DTC defects observed in lin-17+Wnt compound mutants as described above, our previous findings demonstrated that quintuple Wnt mutants, where all five Wnt genes are mutated, exhibit normal DTC production (Yamamoto et al., 2011). This suggests that LIN-17 alone is sufficient for DTC production in the absence of Wnts and that LIN-17’s function is Wnt-independent. To confirm the Wnt-independent role of LIN-17, we constructed LIN-17 with a deletion of the CRD, which is the Wnt binding domain, and expressed it under the lin-17 promoter (osIs113; ΔCRD-LIN-17). As expected, the missing DTC phenotype of lin-17; cwn-2 and lin-17 mom-5/Frizzled was completely rescued by osIs113 (Table 1). Consistently, lin-17 with missense mutations of the conserved cysteine residues of CRD (os2 and mn589) combined with the mom-5 mutation showed only minor, if any, DTC defects (Table 1), in contrast to the complete loss of DTCs in lin-17(n3091) mom-5. These results strongly support the conclusion that LIN-17 regulates the DTC production in a Wnt-independent manner.

The results also suggest that MOM-5/Frizzled might be the receptor for Wnts regulating DTC production, as lin-17 mom-5 double mutants completely lack DTCs. Combining mutations in the other known Wnt receptor genes (mig-1/Frizzled, cfz-2/Frizzled, cam-1/Ror, and lin-18/Derailed) with that of lin-17 did not significantly affect the gonadal phenotype (Table 1). Additionally, mom-5 combined with triple Wnt mutations did not induce the missing DTC phenotype in the absence of lin-17 mutations. While other receptors might also play some roles, MOM-5 appears to be a major receptor for Wnts regulating DTC production.

It has been reported that the absence of DTCs in mutants of intracellular components of the Wnt signaling pathway (Wnt/β-catenin asymmetry pathway) is caused by the loss of polarity of SGPs (Z1 and Z4) which is required for their asymmetric divisions (Siegfried et al., 2004). To determine whether compound mutants of lin-17 and Wnt mutations also affect SGP polarity, we analyzed the localization of sys-1p::GFP::POP-1 (qIs74) after SGP divisions (Figures 1 and 3). In the wild-type, POP-1 is preferentially localized to the nuclei of proximal SGP daughters (Z1.p and Z4.a) compared to the distal ones (Z1.a and Z4.p), representing SGP polarity (LH and HL polarity in terms of sys-1p::GFP::POP-1 localizations for Z1 and Z4, respectively). These asymmetries were strongly disrupted and weakly affected in lin-17 mom-5 double and lin-17 single mutants, respectively, as described previously (Phillips et al., 2007; Siegfried et al., 2004). In contrast, our previous finding demonstrated that SGP polarity was normal in quintuple Wnt mutants (lin-44; cwn-1; egl-20 cwn-2; mom-2) (Yamamoto et al., 2011).

To evaluate SGP polarity using sys-1p::GFP::POP-1, we first quantified the ratios of signal intensities (on a logarithmic scale) of sys-1p::GFP (NLS) which localizes symmetrically between the daughter cells, and calculated the 95% confidence interval (CI) for symmetrically localized signals. Then, we quantified the ratios (on a logarithmic scale) of sys-1p::GFP::POP-1 signal intensities proximal to distal daughter cells in various genotypes (Figure 3A and Figure 3—figure supplement 1). Values within the 95% CI (between the red lines in Figure 3B) indicate symmetric localization, while values lower than the 95% CI (below the lower red line) indicate reversed localization, respectively. Most compound mutants containing lin-17 and cwn-2 mutations exhibited symmetric localization except for lin-17; egl-20 cwn-2, consistent with their missing DTC phenotype (Table 1). The results strongly suggest that the missing DTC phenotype of these mutants is caused by symmetric divisions of SGPs. On the other hand, lin-17; egl-20 cwn-2 mutants that exhibited the Dpd phenotype showed reversed Z1 polarity, suggesting a possible link between the polarity reversal of Z1 and the Dpd phenotype (see below).

To understand the effects of each mutation on SGP polarity, we next statistically compared the distributions of the POP-1 signal ratios among the compound mutants (statistical analyses in this chapter were done by Student’s t-test). To evaluate the effects on SGP polarization irrespective of orientation, we compared the absolute ratios (on a logarithmic scale) of signal intensities between proximal and distal daughter cells (Figure 3—figure supplement 2) rather than the signed ratios described in Figure 3B. Symmetric POP-1 localization in the lin-17 mutant was strongly and moderately enhanced by the cwn-2 mutation for the Z1 and Z4 cells, respectively (p<0.0001 for Z1 and p=0.0108 for Z4 at 15°C; p=0.1400 for Z1 and p=0.4764 for Z4 at 22.5°C in comparison of absolute differences). In the lin-17; cwn-2 background, this symmetry was further enhanced by the cwn-1 mutation for the Z4 cell (Figure 3—figure supplement 2: p=0.0995 at 15°C; p=0.0088 at 22.5°C in comparison of absolute differences), indicating that, in the absence of LIN-17, Z1 polarity is mostly controlled by CWN-2, while Z4 polarity is regulated by both CWN-1 and CWN-2. In contrast to the effects of cwn-1, the egl-20 mutation rescued the polarization defect in the lin-17; cwn-2 background at least at 15°C (p<0.0001 for Z1 and p=0.0007 for Z4 in comparison of absolute differences), indicating that egl-20 has a negative role for SGP polarization. SGP polarization in lin-17; egl-20 cwn-2 appears to depend on cwn-1 at 22.5°C (p=0.0592 for Z1 and p<0.0001 for Z4 in comparison of absolute differences).

In contrast to the rescue of polarization by egl-20 in the lin-17 cwn-2 background, comparison of signed but not absolute differences (Figure 3B) showed it causes reversal of Z1 but not Z4 polarity (p<0.0001 at 15°C and p=0.0015 at 22.5°C). Interestingly, in this triple mutant, especially at 15°C, Z1 and Z4 have the same HL polarity orientation, indicating that the mirror symmetry of SGP polarity is established through the functions of lin-17, egl-20, and cwn-2.

Although the egl-20 mutation suppressed the loss of polarity phenotype in the lin-17; cwn-2 background, it did not in the lin-17; cwn-1 cwn-2 background (p=0.6327 and 0.5741 for symmetric Z1 and Z4 polarity, respectively, between lin-17; cwn-1; cwn-2 and lin-17; cwn-1; egl-20 cwn-2 in comparison of absolute differences at 22.5°C: Figure 3—figure supplement 2), suggesting that egl-20 represses cwn-1 function. One possible explanation may be that egl-20 suppresses the expression of cwn-1. However, the expression levels of cwn-1p::CWN-1::GFP (Yamamoto et al., 2011) are similar among wild-type, lin-17; cwn-2, and lin-17; egl-20 cwn-2 animals (Figure 3—figure supplement 3).

To further confirm the defects in SGP polarity in the compound mutants, we also analyzed the localization of SYS-1/β-catenin (Figure 3C and D) which is regulated independently of POP-1 localization (Phillips et al., 2007). In both wild-type and lin-17 single mutants, Venus::SYS-1 is preferentially localized to the distal SGP daughters than the proximal ones (Phillips et al., 2007). Consistent with the defects of POP-1 localization, we found that lin-17; cwn-2 exhibited symmetric SYS-1 localization for Z1 and less frequently for Z4, while it was reversed for Z1 in lin-17; egl-20 cwn-2 (Figure 3C and D). These results demonstrated that SGP polarity is regulated by the parallel functions of LIN-17/Fz and Wnts.

To understand how SGP polarity orientation is regulated, we examined whether Wnt functions are instructive or permissive. We attempted to reverse Wnt gradients by ectopically expressing CWN-1 and CWN-2 which are normally expressed posteriorly and anteriorly relative to the gonad, respectively (Harterink et al., 2011). We utilized osEx395 (ceh-22p::CWN-1::Venus expressed in the pharynx) and osEx402 (egl-20p::CWN-2::Venus expressed near the anus) which have been demonstrated to provide weak and strong rescue, respectively, of polarity defects in seam cells in the cwn-1; egl-20 cwn-2 background (Yamamoto et al., 2011). However, these extrachromosomal arrays failed to significantly affect the production of DTCs in lin-17; cwn-1; egl-20 cwn-2 (instead of osEx402, its integrant osIs93 was used) (Table 1) nor mis-migration of DTCs in lin-17; egl-20 cwn-2 for osEx402 (Figure 2). Therefore, it remains unclear whether these Wnts are instructive or permissive for SGP polarity.

In the wild-type, the anterior and posterior DTCs are born and migrate on the right-anterior and left-posterior sides of the body, respectively. In lin-17; egl-20 cwn-2 mutants with the Dpd phenotype at the early L3 stage, the more posterior DTC among the two DTCs was observed on the left side (left-DTC) at the tip of the gonadal arm similar to the posterior DTC in the wild-type. Meanwhile, the more anterior DTC was on the right side (right-DTC) at the end of the right side gonad. The cup shape of this right-DTC with the reversed orientation compared to that in the right-anterior DTC in wild-type suggests that it was migrating toward the posterior side (Figure 2B). At the late L3 and early L4 stages, both left- and right-DTCs were present in the posterior region. These results strongly suggest that the Dpd phenotype is caused by the reversed migration of the DTC derived from Z1.

How are the migratory directions of DTCs controlled? Based on Z1 polarity reversal and the Dpd phenotype in lin-17; egl-20 cwn-2 mutants, we initially examined the possibility that the direction of DTC migration correlates with the polarity of the DTC mother cell (DTC is a granddaughter of Z1/Z4) and that the Dpd phenotype is caused by the polarity reversal of the DTC mother. Since Z1 polarity is completely reversed in lin-17; egl-20 cwn-2 at 15°C, the DTC is likely to be produced from Z1.p rather than Z1.a. To understand the relationship between Z1.p polarity and the directions of DTC migration derived from Z1.p, we first examined Z1.p polarity by GFP::POP-1 asymmetry between its daughter cells, and then observed DTC positions after the recovery and growth of the animals. In the observed triple mutants, Z1.p polarity was LH (n=5) and HL (n=5) polarity, indicating that Z1.pa and Z1.pp, respectively, became DTCs. In both cases, the DTC migrated either anteriorly or posteriorly (Figure 4A), suggesting that the migratory direction is not correlated with Z1.p polarity.

The results suggest that DTCs produced at the position of Z1.pa or Z1.pp can migrate either anteriorly or posteriorly. To further confirm this, we identified DTCs after the Z1.p division using lag-2::GFP expression instead of GFP::POP-1. In the wild-type, the lag-2 promoter::GFP is weakly expressed in the Z1/Z4 cell and its expression becomes stronger in Z1.aa/Z4.pp (DTC) soon after they are born (Fujita et al., 2007; Henderson et al., 1994). To unambiguously identify DTCs in lin-17; egl-20 cwn-2 mutants, we expressed the photo-convertible fluorescent protein mKikGR under the lag-2 promoter, converted it to red in Z1.p, and then, after the recovery and growth of the animals until the Z1.p division, we identified DTCs by newly synthesized green mKikGR. As shown in Figure 4B, when Z1.pp became a DTC, it migrated either anteriorly or posteriorly. Although we observed only one animal in which Z1.pa became a DTC, this animal showed the Dpd phenotype indicating posterior migration. Together with the experiments using GFP::POP-1 described above, the results demonstrated that when Z1.pa and Z1.pp become DTCs, they can migrate either anteriorly or posteriorly.

The results raised the possibility that ectopic positions of DTCs inside the gonad rather than at the distal ends in lin-17; egl-20 cwn-2 mutants cause abnormal migration. If so, when Z1.aa (canonical DTC) becomes a DTC in the triple mutants, it should migrate anteriorly. To examine this, we isolated Z1.aa by sequential ablation of Z1.p and Z1.ap in lin-17; egl-20 cwn-2 animals grown at 22.5°C. Since the reversal of Z1 polarity is partial at 22.5°C (Figure 3B), we expected that Z1.aa could become a DTC even in the triple mutants. However, all 10 animals we examined had one posterior gonadal arm, suggesting that Z1.aa rarely becomes DTC in the triple mutants (Figure 4C). In contrast, when Z1.ap was isolated by sequential ablation of Z1.p and Z1.aa, 3 out of 15 animals had a normal anterior arm, and 3 out of 15 showed the Dpd phenotype. Taken together, ectopic DTC produced at the positions of Z1.ap, Z1.pa, and Z1.pp migrate randomly, either anteriorly or posteriorly, at least in lin-17; egl-20 cwn-2 animals.

These results suggest two possibilities explaining abnormal DTC migration in lin-17; egl-20 cwn-2. Ectopically positioned DTCs can migrate randomly irrespective of Wnt signaling mutations. Alternatively, migration of ectopic DTCs in addition to SGP polarity is regulated by Wnt signaling, and abnormal migration occurs only in the absence of some or all of lin-17, egl-20, or cwn-2 functions. To distinguish these possibilities, we used a genetic background that produces ectopic DTCs without mutations in Wnt signaling genes. It was reported that RNAi of cki-1 encoding a cyclin inhibitor causes ectopic DTCs (Kostić et al., 2003). However, the migratory direction of such ectopic DTCs was not examined. We confirmed that cki-1(RNAi) causes ectopic DTCs in addition to the loss of DTCs (Figure 5A). To determine the migratory direction of Z1-derived ectopic DTCs, we ablated Z4 in cki-1(RNAi) animals. In 7 out of 22 animals, we observed posteriorly migrated DTCs (Figure 5B and C). The results show that ectopic DTCs from Z1 can migrate either anteriorly or posteriorly irrespective of mutations in the Wnt signaling genes.

It has been reported that DTCs are not self-propelled but rather pushed distally by proliferating germ cells (Agarwal et al., 2022). However, the posterior migration of DTCs produced from Z1 in lin-17; egl-20 cwn-2 mutants and Z4-ablated cki-1(RNAi) animals cannot be explained by the pushing mechanism of germ cells. Even in such animals, Z1-derived DTCs should be born at the anterior side of germ cells, and some of them migrate posteriorly. At the late L2 stage of lin-17; egl-20 cwn-2 animals, differentiated DTCs expressing mig-24::Venus can be observed in the center of the gonad surrounded by germ cells expressing the germ cell marker xnSi1 in addition to Z4-derived DTC at the posterior end of the gonad (Figure 6A and B). After the recovery and growth of such animals, we found animals with the Dpd phenotype (2/5 and 3/4 without and with a germ cell marker xnSi1, respectively), showing that Z1-derived DTCs migrate through but are not pushed by germ cells. In addition, among mes-1 animals that lack germ cells, we found 84% of DTCs (n=90) were observed outside of the central region near the developing vulva at the L3-L4 stages, suggesting their distal migration to some extent (Figure 6C–E). We even found one animal (n=45) whose DTCs apparently underwent dorsal turn judged by the outline of the gonad marked by lam-1::mCherry (Figure 6C). The results show that DTCs have the ability to migrate independently of germ cells. We noticed that some mes-1 animals that lack germ cells have both of the two DTCs on the anterior side (2/45) or on the posterior side (5/45) (Figure 6D). This may be caused by polarity reversal of SGP (Yamamoto et al., 2011) or DTC mother cells placing DTCs at ectopic positions in mes-1 mutants.

We have previously shown that the polarity of seam cells (V1-V5) is redundantly regulated by cwn-1, cwn-2, and egl-20 (Yamamoto et al., 2011), indicating that all three of these Wnts induce the same polarity orientation (HL for high-low POP-1 concentration). In contrast, we show that these Wnts have distinct functions for SGP polarity at least in the lin-17 mutant background (Figure 7). CWN-1 induces HL polarity for both Z1 and Z4. CWN-2 induces the opposite polarity; LH for Z1 and HL for Z4. EGL-20 inhibits the polarization of both Z1 and Z4. The distinct responses of Z1 and Z4 to CWN-2 appear to be a key for their opposite polarity orientation and mirror-symmetric gonadogenesis. Since these cells have asymmetry in terms of their contacts with germ cells on their proximal sides and those with the basement membrane on their distal sides, CWN-2 may permissively facilitate this asymmetry to control POP-1 localization. Alternatively, Z1 and Z4 may have intrinsic differences in gene expression, leading to distinct responses to CWN-2. The function of CWN-1 for SGP polarity appears to be the same as for seam cell polarity, inducing HL polarity in both cases. Since it was suggested that CWN-1 is a permissive signal for seam cell polarity (Yamamoto et al., 2011), it may also permissively regulate SGP polarity.

In contrast to the role of EGL-20 in seam cells, where it induces HL polarity, it inhibits cell polarization in SGPs. The loss of SGP polarity phenotype in the lin-17; cwn-2 double mutant but not in the lin-17; cwn-1 cwn-2 triple mutants was suppressed by further mutating egl-20. The recovered polarity in lin-17; egl-20 cwn-2 depends on cwn-1, as lin-17; cwn-1; egl-20 cwn-2 mutants showed a strong loss of polarity phenotype. However, cwn-1 cannot efficiently polarize SGPs in lin-17; cwn-2 mutants with normal egl-20 function. Therefore, egl-20 appears to inhibit the cwn-1 function but not its expression (Figure 3—figure supplement 2). A similar antagonistic relationship between cwn-1 and egl-20 was reported for later DTC migration when DTCs turn to migrate centrally on the dorsal side (phase III) (Levy-Strumpf and Culotti, 2014). egl-20 mutants show a reversal of phase III migration and this phenotype is suppressed by the cwn-1 mutation. Further studies are necessary to elucidate the distinct functions of CWN-1, CWN-2, and EGL-20.

We have shown that lin-17/Fzd functions in a Wnt-independent manner to control SGP polarity, since the missing DTC phenotype of lin-17; cwn-2 and lin-17 mom-5 was completely rescued by ΔCRD-LIN-17. In addition, SGP polarity is normal in the quintuple Wnt mutant that has mutations in all the Wnt genes (Yamamoto et al., 2011). In seam cells, Wnt receptors including LIN-17/Fzd and MOM-5/Fzd appear to have Wnt-independent functions for cell polarization, as seam cells are still mostly polarized in the quintuple Wnt mutants, while they are strongly unpolarized in the triple receptor mutants (lin-17 mom-5 cam-1/Ror) (Yamamoto et al., 2011). In Drosophila, Fz/Fzd has been primarily considered to function Wnt-independently to coordinate PCP between neighboring cells (Lawrence et al., 2007), though Fz function can still be regulated by Wnt, as PCP orientation can be directed by ectopically expressed Wnt proteins (Wu et al., 2013).

In Drosophila, Fz regulates PCP by interacting with other PCP components including Van Gogh (Vang). In C. elegans, we found that vang-1/Vang does not appear to function with LIN-17/Fz, since most vang-1 single mutants and cwn-1 cwn-2 vang-1 triple mutants have two gonadal arms (215/216 and 58/58, respectively). As Fz interacts with Disheveled (DSH) in Drosophila PCP regulation, in C. elegans, the Disheveled homologs DSH-2 and MIG-5 regulate SGP polarity (Phillips et al., 2007). Therefore, LIN-17 might regulate the DSH homologs in a Wnt-independent manner.

We have shown that Z1-derived DTC can migrate posteriorly in cki-1(RNAi) as well as lin-17; egl-20 cwn-2 animals, indicating that ectopically positioned DTCs can mis-migrate passing through germ cells irrespective of mutations in Wnt signaling genes. The results strongly suggest that DTCs at ectopic positions can self-migrate to the distal ends of the gonad, regardless of the pushing forces by germ cell proliferation. After reaching the distal ends, further distal migration is likely guided by germ cell proliferation and the degradation of the basement membrane at the distal sides (Agarwal et al., 2022). However, the self-migration of ectopic DTCs to the distal ends suggests that such migratory ability of DTCs may help their further distal migration.

While we were unable to observe migration of Z1.aa-derived DTC in the triple mutants, it is essential to note that Z1.aa-derived DTCs consistently migrate anteriorly in wild-type animals and likely in other genetic backgrounds, giving the absence of the Dpd phenotype in previous reports. Consequently, the regulation of SGP polarity by Wnt signaling plays a crucial role in ensuring normal DTC migration and mirror-symmetric organogenesis by positioning DTCs at the distal edge of the gonad.

N2 Bristol was used as the wild-type strain (Brenner, 1974). The following mutations and transgenes were used: cwn-1(ok546) (deletion) (Zinovyeva and Forrester, 2005), cwn-2(ok895) (deletion) (Zinovyeva and Forrester, 2005), egl-20(n585) (missense behaving like null) (Maloof et al., 1999), lin-17(n3091) (nonsense) and lin-17(n671) (nonsense) (Sawa et al., 1996), mig-1(e1787) (nonsense) (Pan et al., 2006), mom-5(ne12) (nonsense) (Rocheleau et al., 1997), cam-1(gm122) (nonsense) (Forrester et al., 1999), cfz-2(ok1201) (deletion) (Zinovyeva and Forrester, 2005), lin-18(e620) (nonsense) (Inoue et al., 2004), osIs113 (ΔCRD-LIN-17); osEx395 (ceh-22p::CWN-1::VENUS) (Yamamoto et al., 2011), osIs93 (egl-20p::CWN-2::VENUS), a spontaneous integration of osEx402 (Yamamoto et al., 2011), tkIs12 (mig-24::Venus) (Tamai and Nishiwaki, 2007), qIs74 (sys-1p::GFP::POP-1) (Siegfried et al., 2004), qIs95 (VENUS::SYS-1) (Phillips et al., 2007), qyIs127 (lam-1::mCherry) (Ihara et al., 2011), xnSi1 (mex-5::GFP::PH) (Chihara and Nance, 2012). cki-1(RNAi) was performed as described previously (Fujita et al., 2007). lin-17(os2) was identified in a screen for the Psa (phasmid socket absent) phenotype (Sawa et al., 2000). lin-17(os2) and lin-17(mn589) (gifted by Mike Herman) carry mutations in the second and seventh cysteine residues of the CRD domain (C36Y and C104Y), respectively. os2 and mn589 exhibit 38% and 47% Psa phenotype (indicating T cell polarity defects), respectively, while lin-17(n3091) shows 95% Psa phenotype (Goldstein et al., 2006). mes-1(bn7) is a temperature-sensitive allele with higher penetrance of the germless phenotype at 25°C than at 15°C, and was grown at 22.5°C. The germless phenotype of mes-1(bn7) was observed by the absence of the mex-5::GFP::PH signal through direct observation of epifluorescence.

ΔCRD-LIN-17 (pMM39) contains a 5 kb XhoI genomic fragment upstream of the lin-17 start codon and a lin-17 cDNA fused to Venus at its C-terminus in the pPD49.26 vector (gift of A Fire). The CRD domain was precisely deleted from the lin-17 cDNA. sys-1p::GFP(NLS) (pSS20) contains the sys-1 promoter from the GFP-POP-1 plasmid (pJK707) (Siegfried et al., 2004) inserted into pPD95.67 (gift of A Fire). lag-2p::NLS::mKikGR (pSN17.4) contains a 7.4 kb lag-2 promoter fragment and NLS::mKikGR coding sequence (codon-optimized). pMM39, pSS20, pSN17.4, and mig-24::Venus (Tamai and Nishiwaki, 2007) plasmids were injected into unc-76(e911) animals along with the Unc-76 rescuing plasmid (Bloom and Horvitz, 1997) to obtain osEx576, osEx443, osEx509, and osEx283, respectively. Subsequently, osEx576 and osEx509 were integrated by UV-irradiation to generate osIs113 and osIs168, respectively.

Localization of POP-1 and SYS-1 was observed using confocal microscopy (Zeiss LSM700) and signal intensities were quantified with ImageJ software. As SGP daughter cells are situated in distinct focal planes, the signal ratios of GFP::POP-1 (sys-1p::GFP::POP-1) [qIs74] between SGP daughter cells were normalized using the levels of sys-1p::GFP(NLS) [osEx443], which are considered to be the same between them. First, in wild-type animals with osEx443 but not qIs74, signal intensities in the SGP daughter cells at focal planes showing maximum intensity in each cell were quantified. Distances (Z1.p-Z1.a or Z4.a-Z4.p) along the Z axis were also recorded. Ratios of signal intensities (Z1.p/Z1.a or Z4.a/Z4.p) on a logarithmic scale and distances were plotted on the Y axis and X axis, respectively.

Using these plots, a regression line was calculated:y=−0.034x+0.0148

To assess the variability of the predicted values, we calculated the predicted values of the regression line and their CI. First, the standard error (SE) of the residuals was determined, which was 0.0613. Next, given a sample size of 97, the degrees of freedom were 95. Based on these degrees of freedom, the t-value for a 95% CI was calculated. Finally, the upper (e_upper) and lower (e_lower) bounds of the the 95% CI for each predicted value ŷ were calculated using the following formulas:eupper=y^+t×SEelower=y^−t×SE

The deviations of the upper and lower bounds of the 95% confidence interval were defined as follows:υupper=eupper−y^υlower=elower−y^

Subsequently, distances (Z1.p-Z1.a or Z4.a-Z4.p) along the Z axis as well as fluorescent intensities in both wild-type and mutant animals with sys-1p::GFP::POP-1 (qIs74) were recorded, and the distances were used as the x values in the regression equation to calculate theoretical ratios of fluorescence expressed equally between SGP daughter cells. Then, the observed fluorescence ratios of sys-1p::GFP::POP-1 on a logarithmic scale were adjusted by subtracting these theoretical values calculated from the distances (ri).ri=yi−y^i

Finally, the absolute and signed values of the results (absolute and signed differences) were used to evaluate extent and orientation of SGP polarity, respectively. To evaluate the absolute differences, cases where the residuals were greater than v_upper were assessed as exhibiting polarity formation (ri>v_upper). Conversely, for the signed differences, cases where the residuals were greater than v_upper were defined as having normal polarity(ri>v_upper), and cases where the residuals were less than v_lower were defined as having reversed polarity (ri<v_lower).

The violin plot was created using the Seaborn library in Python. For absolute differences (|ri| values were used), the parameter cut=0 was utilized in violin plots to truncate the kernel density estimation at the minimum and maximum data points, ensuring that the density tails did not extend beyond the actual data range. In contrast, for signed differences (ri values were used), the parameter cut=2 was used, allowing the kernel density estimation to extend beyond the observed data range, providing a smoother and more comprehensive visualization of the data distribution. Additionally, to maintain a consistent bandwidth for kernel density estimation in the violin plots, the parameter bw=0.2 was set for all plots. v_upper and v_lower were used as the 95% CI on the violin plot. We used the Student’s t-test to compare the differences in fluorescence intensities between the two groups of interest. The statistical analysis was performed using JMP9 software (SAS, USA).

The presence, absence, and positions of DTCs were determined by direct observation of the fluorescence of mig-24::Venus using epifluorescent microscopy. For animals lacking mig-24::Venus, the assessment was made based on gonad arms under Nomarski microscopy for animals that do not have mig-24::Venus. These observation was performed at the L4 or young adult stages. Initial DTC migratory direction was judged by considering the position and cup shape of the DTCs. Anterior and posterior positions of DTCs indicated initial anterior and posterior migration, respectively. When DTCs were observed in the center, their migratory direction (which is opposite to the initial migratory direction) was judged by assessing their cup shape.

The animals were immobilized on slide glasses using 10 mM sodium azide. Laser ablation was performed by a MicroPoint system (Photonic Instruments) equipped with a 2 mW pulsed nitrogen laser (model VL-337; Laser Science Inc) exciting Coumarin 440 dye. Photoconversion was performed through irradiation with 405 nm laser on Zeiss LSM510 confocal microscope.