The Wnt receptor CAM-1 prevents axons from crossing the midline

Proper axon guidance requires the synergy of different signaling pathways. The Netrin and Robo pathways have previously been implicated in the midline guidance of C. elegans VNC axons (Ishii et al. 1992; Keleman et al. 2002), but it is unclear whether other pathways are involved. We decided to examine whether the Wnt pathway is implicated in midline guidance in C. elegans and first studied whether loss of the Wnt receptor CAM-1 caused crossover defects of VNC axons. cam-1 encodes a receptor tyrosine kinase belonging to the Ror family that is thought to bind Wnt glycoproteins via its cysteine rich domain (CRD) (Forrester et al. 2004). Almost 80% of the PVQ axons and 70% of the HSN axons cross over from the left to the right side in cam-1(gm122) null mutants. The PVP neurons normally cross the midline only once (Figure 1A), but in cam-1 mutants, the left axon crossed over at one additional site in >80% of the worms. Most often the axons crossed the midline at various locations in the middle region of the worm (Figure 1B). Interestingly, theses PVP and PVQ defects were already present in newly hatched L1 larvas (data not shown), suggesting that the defects are developmental and not maintenance in nature. Previous studies suggest that the PVP and PVQ axons serve as substrates for the HSN axons in the VNC (Garriga et al. 1993), and HSN guidance defects likely depend on earlier guidance errors of PVP and PVQ. We decided therefore to focus the remaining studies on the PVP and PVQ axons.

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Figure 1

CAM-1 prevents axons in the VNC from inappropriately crossing the midline. (A) Diagram of a wild-type worm showing the anatomy of PVPL/R, PVQL/R, and HSNL/R neurons and axons. The PVP and PVQ neurons have their cell bodies in the tail and extend axons anteriorly to the nerve ring in the head. The HSN neurons are located slightly posterior to the vulva in the midbody and project axons ventrally to the midline where they join the VNC. In wild-type worms, the PVP axons cross the midline posterior to the PVT neuron shortly after they have projected their axons, while PVQ and HSN axons do not cross. In the young adults when the scoring was performed, the left and right axons of PVPs and PVQs are actually very close to each other and only begin to separate a quarter of the length between the vulva and the tail (see Figure 7A). This diagram aims to highlight the fact that PVP axons cross over in the tail. AVG and PVT neurons are guidepost cells in the VNC. (B) The anatomy of the HSN and PVQ neurons in a wild-type worm. The HSNs express GFP and the PVQs express mCherry. (C) The anatomy of the PVPs (blue) and PVQs (red) in a wild-type animal. (D) Diagram of representative midline guidance phenotypes in a cam-1(gm122) null mutant, with the PVP, PVQ, and the HSN axons crossing over at locations between the head and the vulva. (E and F) PVP, PVQ, and HSN axons in the left tract cross over to the right side (the site of crossover is indicated by the white arrows). (G) Quantification of crossover defects of PVP, PVQ, and HSN axons in cam-1(gm122) mutants. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. Ventral view, anterior is to the left. The vulva is marked with an asterisk. * P = 0.01–0.05, ** P < 0.01 (Fisher’s exact test). [Psra-6::mCherry; Ptph-1::GFP] was used for the visualization of PVQs (mCherry) and HSNs (GFP) in B and E and hdIs26[Podr-2::CFP; Psra-6::dsRed2] was used to visualize PVPs (CFP) and PVQs (dsRed) in C and F.

A detailed analysis of different cam-1 alleles revealed that the severity of crossover phenotypes correlated with the strength of the alleles. The null allele gm122 is a nonsense mutation that introduces a premature stop codon in the middle of the Wnt binding CRD domain (Figure 2A). The sa692 allele is a missense mutation that changes a conserved cysteine within the CRD domain to tyrosine, whereas ak37 has a large deletion, removing sequences from the kringle domain to the C terminus (Figure 2A). These three alleles exhibited highly penetrant midline guidance defects (Figure 2, B and C). The cw82 allele is a missense mutation in a conserved residue in the signaling kinase domain, and ks52 is a deletion allele that removes the kinase domain (Figure 2A). These two alleles had a milder phenotype compared to the other alleles (Figure 2, B and C).

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Figure 2

All five cam-1 alleles exhibit PVP and PVQ crossover defects. (A) cam-1 coding sequence with five cam-1 alleles annotated below. CAM-1 is a transmembrane protein with three extracellular domains [i.e., immunoglobulin (Ig) domain, cysteine-rich domain (CRD), and Kringle domain (Kr)] and two intracellular domains [i.e., kinase domain and serine/threonine (S/T)-rich domain]. The null allele gm122 is a nonsense mutation that introduces a premature stop codon in the middle of the Wnt binding CRD domain. The sa692 allele is a missense mutation that changes a conserved cysteine within the CRD domain to tyrosine, whereas ak37 has a large deletion, removing sequences from the kringle domain to the C terminus. The cw82 allele is a missense mutation in a conserved residue in the kinase domain and ks52 is a deletion allele that removes the kinase domain. (B and C) Percentage of PVP and PVQ crossovers in different cam-1 alleles. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. ** P < 0.01 (Fisher’s exact test).

The CWN-1, CWN-2, and EGL-20 Wnts, the LIN-17 Wnt receptor, and the PCP protein VANG-1 also regulate midline guidance

Next, we asked whether other Wnt signaling genes are involved in midline guidance. There are five Wnt genes in C. elegans (cwn-1, cwn-2, egl-20, lin-44, and mom-2) (Shackleford et al. 1993; Herman et al. 1995; Maloof et al. 1999) and four Frizzled receptor genes (cfz-2, lin-17, mig-1, and mom-5) (Sawa et al. 1996; Rocheleau et al. 1997; Ruvkun and Hobert 1998). In addition, there is a single ortholog of Ryk, a receptor tyrosine kinase-like molecule with an extracellular Wnt-binding Wnt-inhibitory factor (WIF) domain (Inoue et al. 2004). We first asked whether PVP and PVQ crossed the midline inappropriately in four of the single Wnt mutants (cwn-1, cwn-2, egl-20, and lin-44). MOM-2 specifies endodermal cell fates and the null mutant is lethal (Rocheleau et al. 1997). Very few mom-2 homozygous progeny produced from mom-2 heterozygous animals survived to the scoring stage of young adult (n = 10 with PVP crossover defect of 10% and PVQ crossover defect of 10%), precluding us from further analyzing the role of mom-2 in midline guidance. Only loss of cwn-2 caused crossover defects that were significantly different from wild-type worms (Figure 3). Next, we created double and triple mutant combinations with cwn-1, cwn-2, and egl-20 and found that there was a significant increase of crossovers in the cwn-1; cwn-2 double mutant and in the cwn-1; cwn-2; egl-20 triple mutant, where >80% of PVP and PVQ exhibited crossover defects (Figure 3). This result indicates that CWN-1, CWN-2, and EGL-20 act together to prevent VNC axons from crossing the midline. We also studied the ortholog of Drosophila and human Wntless, mig-14, which is required for secretion of Wnts (Pan et al. 2008). We only observed mild PVP and PVQ defects (∼30%) in mig-14(ga62) mutants, indicating that there is still enough Wnts secreted in this hypomorphic allele to prevent erroneous crossovers (Figure 3).

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Figure 3

The Wnts CWN-1, CWN-2, EGL-20, and the Wnt receptor LIN-17 regulate midline guidance. Quantification of crossover defects of PVP (A) and PVQ (B) in single, double, and triple mutant combinations of the Wnt mutants cwn-1, cwn-2, egl-20, and lin-44, the Wntless homolog mutant mig-14, and the Wnt receptor mutants cfz-2, lin-17, mig-1, and mom-5. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. Single mutants were compared with wild type, double, and triple mutants with the single mutants combined. * P = 0.01–0.05, ** P < 0.01 (Fisher’s exact test).

Next, we studied the Wnt receptors and found that only mutations in one of the four Frizzled genes, lin-17, caused significant defects, where 40% of the PVP and PVQ axons crossed the midline. To test whether cam-1 and lin-17 function in the same or in parallel pathways, we created a lin-17; cam-1 double mutant. This mutant exhibited the same penetrance of defects as the cam-1 single mutant (Figure 3), indicating that they function in the same pathway. Furthermore, a mutation in cwn-2 failed to enhance the midline phenotype in either a lin-17 or lin-44 mutant, suggesting that these genes work in the same pathway (Figure 3).

Axon pathfinding is primarily mediated by the PCP pathway that regulates cytoskeletal dynamics by activating the small GTPases Rho, Rac, and Cdc42 (Schlessinger et al. 2009; Zou 2012). In the PCP pathway, Frizzled receptors associate with Dishevelled phospho proteins, the transmembrane protein Van Gogh/Strabismus, and its adaptor protein Prickle and the atypical cadherin Flamingo/Starry night (Rosso et al. 2005; Zou 2012). The C. elegans Flamingo ortholog FMI-1 was recently shown to have severe VNC guidance defects, where 81% of PVPR crossed over to the right tract and the PVQ axons crossed the midline multiple times (Steimel et al. 2010). We asked whether any of the three C. elegans dishevelled genes (dsh-1, dsh-2, and mig-5), the Van Gogh ortholog vang-1, and the Rac ortholog ced-10 also exhibit midline guidance defects. The single dishevelled mutants displayed only mild defects, indicating that they either function redundantly or are not involved in midline guidance. A more detailed analysis of the overlapping functions of dishevelleds proved difficult, as worm strains containing mutations in two or three dishevelled needed to be kept in a heterozygous state and no homozygous progeny reached the scoring stage of young adult. On the other hand, both vang-1 and ced-10 mutants exhibited significant defects, suggesting that the PCP pathway plays an important role in C. elegans midline guidance (Figure 4). Furthermore, a mutation in vang-1 failed to enhance the crossover phenotype in either a cam-1 or lin-17 mutant (Figure 4), consistent with the idea that VANG-1 works together with CAM-1 and LIN-17 in midline guidance.

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Figure 4

PVP and PVQ crossover defects in mutants in the Wnt PCP pathway. Quantification of crossover defects of PVP (A) and PVQ (B) in the Wnt PCP pathway mutants ced-10, dsh-1, dsh-2, mig-5, and vang-1. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. ** P < 0.01 (Fisher’s exact test).

CAM-1, LIN-17, and VANG-1 function nonautonomously in PVT to regulate midline guidance of PVP and PVQ

To examine where CAM-1 functions to regulate midline guidance, we first asked whether cam-1 is expressed in PVP and PVQ or in nearby cells where CAM-1 might function nonautonomously. For the cam-1 expression studies, we used a strain that has an integrated full-length Pcam-1::cam-1::GFP construct (a kind gift from Wayne Forrester) (Figure 5B). cam-1 is broadly expressed in many cell types, including muscle, hypodermis, and neurons (Forrester et al. 1999), making direct identification of relevant cells difficult. To determine whether cam-1 is expressed in PVP and PVQ neurons, we crossed the Pcam-1::cam-1::GFP strain with hdIs26[Podr-2::CFP; Psra-6::dsRed2], which expresses CFP in PVP and dsRed in PVQ, and asked whether the different fluorophores colocalized. We detected strong CAM-1 expression in PVQ but only weak expression in PVP (Figure 5, A–C). The Pcam-1::cam-1::GFP transgene rescues the migration defects of ALM, BDU, CAN, and HSN cell bodies in the cam-1(gm122) null mutant (Kim and Forrester 2003), and we found that it also rescues cam-1 midline guidance defects (Figure 5, K and L).

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Figure 5

cam-1 is expressed and functions in the guidepost cell PVT. (A) PVP and PVQ neurons in a L4 larva were visualized with hdIs26[Podr-2::CFP; Psra-6::dsRed2] that expresses CFP in PVPs and dsRed in PVQs. (B) Expression of Pcam-1::cam-1::GFP in the same worm. (C) Overlay of A and B shows that cam-1 is strongly expressed in PVQs and barely detectable in PVPs. (D) oyIs14[Psra-6::GFP]; otIs37[Punc-47(del)::GFP] in a L2 larva shows expression of GFP in PVT and PVQs. (E) Expression of Pcam-1::cam-1::mCherry in the same worm. (F) Overlay of D and E shows that cam-1 is expressed in PVT and PVQs. The images in A–C were acquired with a Zeiss LSM700inv laser scanning confocal microscope with a 20× objective. For images in D–F, we used an Axioscope 2, 40× objective. (G) Expression of lim-6int3::GFP in an embryo at the comma stage. Expression is seen in AVL, DVB, RIS, and PVT (white arrow). (H) Differential interference contrast (DIC) image of the same embryo as in G. (I) lim-6int3_short::GFP is only expressed in PVT (white arrow). (J) DIC image of the same embryo as in I. Bar, 100 μm. Quantification of crossover defects of PVP (K) and PVQ (L) in nontransgenic cam-1(gm122) mutants compared to cam-1 mutants carrying transgenes containing cam-1 cDNA expressed from either its own promoter Pcam-1 (multiple tissue), a hypodermis-specific promoter (Pelt-3), a promoter expressed in PVP (Podr-2), a promoter expressed in PVQ (Psra-6), or from a promoter containing elements that drive expression specifically in PVT (lim-6int_short). Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. At least two independent, nonintegrated transgenic lines were generated for each construct. ** P < 0.01 (Fisher’s exact test).

Next, we examined whether CAM-1 functions autonomously in PVP and PVQ and expressed cam-1 complementary DNA (cDNA) with the same promoters used in the expression study. The odr-2 (Chou et al. 2001) and the sra-6 (Troemel et al. 1995) promoters were used to express cam-1 in PVP and PVQ, respectively. The same promoters were previously used to express flamingo/fmi-1 to rescue the PVP and PVQ crossover defects in fmi-1 mutants (Steimel et al. 2010). To our surprise, neither of the cam-1 constructs rescued the midline phenotype of cam-1 mutants significantly (Figure 5, K and L).

We then decided to express cam-1 in cells located in close proximity to the PVPs and PVQs, such as hypodermal cells and the guidepost cell PVT, which is located on the midline between the two VNC tracts. We used the elt-3 promoter to express cam-1 specifically in the hypodermis (Gilleard et al. 1999), but this construct failed to rescue PVP or PVQ crossovers in cam-1 mutants (Figure 5, K and L). Finally, we examined whether CAM-1 is expressed and functions in PVT. For colocalization studies, we created a mCherry-tagged cam-1 transgene (Pcam-1::cam-1::mCherry) and crossed it with oyIs14[Psra-6::GFP]; otIs37[Punc-47(del)::GFP], which expresses GFP in PVT, PVQ, and other neurons (Figure 5D). Our results show that CAM-1 is expressed in PVT (Figure 5, D–F).

To study whether CAM-1 functions in PVT, we first had to identify a promoter element that could drive expression of cam-1 cDNA specifically in PVT. Hobert et al. (1999) previously studied the regulatory regions of the lim-6 gene and found that the third intron in the gene (lim-6int3::GFP) can drive expression in only four neurons: PVT, DVB, RIS, and AVL (Figure 5G). We fused different parts of the 1.2-kb intron to the GFP gene and identified a 300-bp region, lim-6int3_short, that drove expression only in PVT (Figure 5I). We detected strong GFP expression in PVT in embryos as early as in the comma stage, well before the PVP neurons extend their axons (Figure 5, G–J). Interestingly, a cam-1 cDNA expressed with this PVT-specific promoter element significantly rescued PVP and PVQ crossover defects in cam-1 mutants (Figure 5, K and L), suggesting that CAM-1 functions nonautonomously in the PVT to prevent other axons from crossing the midline.

The PVT neuron runs along the entire length of the animal and we found that the trajectory of the PVT axon appeared normal in cam-1 mutants (data not shown), suggesting that the guidance errors of PVP and PVQ axons do not result from a guidance defect of PVT axon per se. We considered two scenarios of how CAM-1 may function in PVT. One possibility is that CAM-1 expressed from PVT acts as a sink for the Wnts. Alternatively, CAM-1 acts as a signaling receptor in PVT. To distinguish between these two possibilities, we generated a cam-1 transgene that lacks the signaling intracellular domain and expressed it specifically in PVT [PPVT::cam-1(Δintra)]. This truncated CAM-1 transgene failed to rescue cam-1 midline defects, suggesting that CAM-1 signaling activity in PVT is essential for midline guidance (Figure 5, K and L).

Given that CAM-1, LIN-17 and VANG-1 function in the same pathway, we examined whether LIN-17 and VANG-1 also function in PVT. Consistent with this idea, PVT-specific expression of LIN-17 or VANG-1 significantly rescued the midline defects in a lin-17 mutant or a vang-1 mutant, respectively (Figure 5, K and L). However, transgenic expression of LIN-17 in PVT failed to rescue midline defects in a cam-1(gm122) mutant (data not shown), suggesting that LIN-17 and CAM-1 are not functionally interchangeable in PVT.

The Wnt pathway interacts with the Netrin and Robo pathways to regulate midline guidance

We next asked whether the Wnt pathway interacts with the Robo or Netrin pathways that are known to regulate midline guidance. In C. elegans, loss of the Slit homolog slt-1 causes 20% of PVQ axons to cross the midline (Hao et al. 2001), while in mutants that lack the Slit receptor SAX-3, 55% of PVQ and HSN axons cross over (Zallen et al. 1998). The Netrin homolog UNC-6 is expressed and functions during three different phases of development of the VNC. In the first stage, the ventral hypodermis forms an UNC-6 gradient that is crucial for correct positioning of the VNC neuron cell bodies. In the next stage, the midline AVG and PVT neurons express UNC-6 to direct the pioneer axons from the right and left sides of the VNC. In the last stage, neurons in the lateral lumbar ganglion express UNC-6 to organize the later developing VNC axons (Wadsworth and Hedgecock 1996; Ren et al. 1999).

For our genetic interaction studies, we examined null mutants of slt-1, sax-3, unc-6, and its receptor unc-40 and created the following double mutants: cam-1; slt-1, lin-17; slt-1, cam-1; unc-6, unc-40; cam-1, lin-17; unc-6, and lin-17; sax-3. We also tried to make a cam-1; sax-3 double mutant, but failed since it was synthetic lethal. In cam-1, lin-17, and sax-3 single mutants, the PVP and PVQ axons most often crossed over in the midbody, somewhere between the AVG and the PVT neurons, while in unc-6 and unc-40 mutants, the crossovers occurred most frequently in the tail, posterior to the PVT cell body (Figure 6). The cam-1; unc-6 double mutant exhibited significantly higher penetrance of overall crossover defects, and cam-1; unc-40 had a significant increase of crossovers in the tail, suggesting that cam-1 functions in parallel to the Netrin pathway. lin-17; sax-3 and lin-17; unc-6 also had significantly more crossovers and the majority occurred in the tail, which indicate that sax-3 and unc-6 work in parallel to lin-17 in midline guidance (Figure 6). On the other hand, a mutation in slt-1 failed to enhance the crossover phenotype of either a cam-1 or a lin-17 mutant (Figure 6), suggesting that SAX-3 has SLT-1 independent function in midline guidance.

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Figure 6

The Wnt pathway acts in parallel to the Robo and Netrin pathways in midline guidance. Quantification of crossover defects of PVP (A) and PVQ (B) in single and double mutant combinations of the Wnt pathway mutants cam-1 and lin-17, Netrin pathway mutants unc-6 and unc-40, and the Slit pathway mutants slt-1 and sax-3. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), and red bars posterior to PVT. The number of animals scored for each genotype is indicated above each bar. Single mutants were compared with wild type and double mutants with the single mutants combined. The P-values shown on the graph are pairwise comparisons of the total percentage of crossover defects between the single and double mutants. Comparisons of crossover defects in the tail between these genotypes are also highly significant: P < 0.01 between cam-1 and cam-1; unc-6; P < 0.01 between cam-1 and unc-40; cam-1, and P < 0.01 between sax-3 and lin-17; sax-3. * P = 0.01–0.05, ** P < 0.01 (Fisher’s exact test).

Killing the guidepost cell PVT causes crossover defects

Our data and previous studies (Ren et al. 1999) suggest that the PVT neuron signals to axons in the VNC to prevent them from crossing over to the opposite side during their outgrowth. Not only is PVT secreting UNC-6 that guides the PVP and PVQ axons, but our results also show that CAM-1 is expressed and functions as a signaling receptor in PVT.

PVT functions to both establish and maintain proper guidance of VNC axons. Laser ablation of PVT in embryos causes disorganization of the VNC (Ren et al. 1999) and ablation of PVT during the L1 larval stage causes axons to “flip over” to the other tract (Aurelio et al. 2002). The flip-over phenotype is distinct from the crossover phenotype because it occurs when a segment of the axon crosses over to the opposite side and is typically observed postembryonically, suggesting a maintenance defect of the PVT. Since the study performed by Ren et al. (1999) was done on only five embryos, we decided to ablate a larger number of PVT neurons (N = 130) during embryogenesis to study in more detail how PVT prevents erroneous crossovers during the development of the VNC. To kill PVT, we used a genetic ablation technique developed by Chelur and Chalfie (2007). In this system, the human Caspase-3 is expressed as two subunits that are only active when they are associated via the interaction of the antiparallel leucine-zipper domains. We expressed both subunits under the control of the same PVT-specific element that we used for our expression and rescue experiments. The two constructs were injected into the strain otIs37[Punc-47(del)::GFP]; gmIs65[Psra-6::mCherry; Ptph-1::GFP] that expresses mCherry in PVQ and GFP in PVT, HSN, and other neurons. We found that the caspase constructs fail to abolish GFP expression in PVT in all worms: 17% of the worms had no detectable GFP expression, 52% had weak expression, and 31% had normal expression. Since it is unclear whether PVTs in the worms with weak expression are functional or not, we decided to exclude this group of worms from our analysis. We scored the crossovers of the PVQ axons in animals with or without GFP expression in the PVT (Figure 7D). When PVT was present and expressed GFP at high levels, 12% of the PVQ axons crossed over in the tail. When PVT was absent, the penetrance was even higher: 4% crossed over in the head, 38% crossed over in the middle, and 25% crossed over in the tail. We also noticed that in 8% of worms where the PVT was absent, the PVQ axons flipped over instead of crossed over, which indicates that the PVT might have been alive and functioning during embryogenesis, but died later in the L1 stage (Figure 7D, see Figure 7, A–C for wild type, crossover, and flipover phenotypes). Consistent with our previous results that CAM-1 is acting in the PVT, caspase ablation of PVT in cam-1(gm122) mutants failed to further enhance the PVQ defects (Figure 7D). By contrast, cam-1; unc-6 double mutants exhibited significantly higher crossover defects compared to worms in which the PVTs were killed (Figure 7D).

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Figure 7

Killing the PVT neuron with human Caspase-3 causes the PVQ axons to exhibit both crossover and flipover defects. (A) Wild-type PVQ axons in young adults are very close to each other and only begin to separate a quarter of the length between the vulva and the tail as indicated by the white arrow. (B) Crossover of PVQ axons in the tail region. Note the lack of the separation between the right and left axons, resulting in fused axons on one side of the VNC. (C) Flipover defect of PVQ axons. A segment of the axon usually flips over to the opposite side posterior to the vulva (as indicated by the yellow arrows). The axons still manage to separate a quarter of the length between the vulva and the tail as indicated by the white arrow. The PVQ neurons in A–C are visualized with oyIs14[Psra-6:: GFP]). (D) Quantification of crossover and flipover defects of PVQ. Human Caspase-3 was expressed specifically in PVT in wild-type worms containing fluorescent markers for PVT and PVQs. The transgenic worms were divided into two groups based on the presence or absence of GFP in PVT and then scored for PVQ crossover or flipover defects. The penetrance of crossovers and flipovers in worms with PVT ablation were compared to wild type, cam-1, unc-6, and cam-1; unc-6 double mutants. Green bars show crossover anterior to the AVG neuron (head), blue bars between the AVG and PVT (middle), red bars posterior to PVT, and yellow bars show flipovers along the VNC. The number of animals scored for each genotype is indicated above each bar. * P = 0.01–0.05, ** P < 0.01 (Fisher’s exact test).

Given that CAM-1 and UNC-6 are both expressed in the PVT, we wondered whether there is a crosstalk between the Wnt and Netrin pathways as was observed in the C. elegans distal tip cell (Levy-Strumpf and Culotti 2014). We tested whether UNC-6 expression is regulated by CAM-1 by generating a PVT::unc-6::gfp transgene that was then crossed into cam-1 mutants. We found that there was no obvious difference of UNC-6 expression in PVT between wild type and cam-1 mutants (data not shown).