The evolutionarily conserved Robo family was first identified in the classic genetic studies of Drosophila CNS axonal midline crossing (Seeger et al., 1993) and belongs to the immunoglobulin (Ig) superfamily. Besides their functions in axon guidance (Dickson and Gilestro, 2006), Robo is also involved in cell migration (Killeen and Sybingco, 2008; Viveiros et al., 2011; Hinck, 2004; Grieshammer et al., 2004), organogenesis (Grieshammer et al., 2004; Mommersteeg et al., 2013), cancer development (Wang et al., 2008), and immune cell regulation (Wu et al., 2001; Branchfield et al., 2016). In these processes Robo functions as the receptor for the guidance cue molecule – Slit. However, mutations of Robo cause abnormalities in Caenorhabditis elegans (Zallen et al., 1998; Hao et al., 2001), zebrafish (Fricke et al., 2001), and cultured mammalian neurons (Hivert et al., 2002; Liu et al., 2004) that have not been observed in Slit mutants, suggesting that there may be evolutionarily conserved Slit-independent aspects of Robo function and regulation.

Glial cells often need to migrate over long distances from their birthplace to appropriate regions where they form functional units with neurons or perform other roles (Gilmour et al., 2002; Jarjour and Kennedy, 2004; Kinrade et al., 2001; Klämbt, 2009). Some guidance molecules including those in the Netrin and Semaphorin families were shown to be involved in glial migration (Kinrade et al., 2001; Jarjour et al., 2003; Sasse and Klämbt, 2016; Spassky et al., 2002; Unni et al., 2012). In Drosophila, CNS-derived glial cells can move along nerves to reach their final position, and during migration the glial expression of ROBO2 receptor is required for preventing glial breakaway from the nerve in a Slit-dependent manner (Sasse and Klämbt, 2016).

Proteolytic modifications play important roles in regulating Ig superfamily proteins. One good example is the Netrin receptor DCC. DCC can be cleaved first at the extracellular region and subsequently at the intracellular domains (Neuhaus-Follini and Bashaw, 2015). The DCC intracellular cleavage fragment can then translocate into the nucleus and equip DCC with transcriptional regulatory function (Neuhaus-Follini and Bashaw, 2015). Similarly, NCAM and EphrinA5 can go through extracellular cleavage to release adhesive interactions and cause cell detachment (Brennaman et al., 2014). Drosophila Robo can be cleaved at the FN3 (Fibronectin type-III) repeats of the extracellular region, and the extracellular cleavage fragment can recruit Son of sevenless (Sos) to mediate Slit-dependent midline repulsion (Coleman et al., 2010). However, in all these cases the cleaved fragments and the function of the receptors highly rely on their canonical ligands. It is still unknown whether the cleavage of Ig superfamily receptors can function in a fashion that is independent of their canonical ligands.

There are in total 56 glia in C. elegans, which include 50 neuroepithelial glia that ensheath sensory neuron receptive endings (Oikonomou and Shaham, 2011; Shaham, 2015). The largest sensory organ of the worm, called the amphid sensilla, is a pair of sensilla composed of 12 neurons and two glial cells each (Ward et al., 1975). These glia, called the amphid sheath (AMsh) and amphid socket (AMso) cells, form channels that ensheath the dendrites of sensory neurons in the amphid sensilla (Oikonomou and Shaham, 2011; Ward et al., 1975; Perkins et al., 1986). Furthermore, these glial cells are vital for the proper functioning of the neurons that they ensheath, where they can modulate neural activity through secreted molecules at the receptive endings and control neuron receptive ending shape (Bacaj et al., 2008; Shaham, 2010; Singhvi et al., 2016). In previous studies we show that the fate determination of C. elegans AMsh glial cells utilizes similar mechanisms as those in mammals (Zhang et al., 2020a). AMsh glia are born close to the nose of C. elegans, where their processes anchor to the nose region while the cell bodies migrate toward the nerve ring (Shaham, 2015; Heiman and Shaham, 2009), and the position of AMsh glia is important for their function (Zhang et al., 2020b). With all these advantages, AMsh glia have emerged as a powerful model to study the potential conserved mechanisms underlying glial development and function.

SAX-3/Robo regulates glial migration in a Slit-independent manner

In an unbiased genetic screen for glial migration, we isolated two loss-of-function alleles of sax-3, the only Robo receptor in C. elegans (Zallen et al., 1998), with defects in AMsh glial migration. To quantify the migration of AMsh, we used the pharyngeal terminal bulb as a reference point and found that AMsh cell bodies reside at the side of and align with the center of pharyngeal terminal bulbs in day 1 (D1) young adults (Figure 1a and b). AMsh glia fail to migrate to their final positions in both sax-3(lf) alleles, and yad10 animals display stronger phenotypes than yad147 animals and show a similar extent of defects as those in the null allele of sax-3 (ky123) (Zallen et al., 1998), suggesting that yad10 is likely a null allele of sax-3 (Figure 1a and b and Figure 1—figure supplement 1a and c). To further characterize the sax-3(lf) phenotype we examined AMsh migration at different developmental stages in a single animal and found that sax-3(lf) animals exhibited migration defects in all post-embryonic stages, suggesting that sax-3 is required for AMsh migration during development (Figure 1—figure supplement 1b).

Figure 1 with 1 supplement see all

Download asset Open asset

Next, we examined the function of the canonical Robo ligand, Slit, in AMsh migration and found that loss-of-function of slt-1(ok255, null allele), the only Slit in C. elegans, did not cause any defects, suggesting that Robo functions in a Slit-independent manner to regulate AMsh migration (Figure 1c). As sax-3 is involved in the regulation of nerve ring position (Zallen et al., 1998; Sasakura et al., 2005), it could be argued that the AMsh migration defect could originate from the anteriorly positioned nerve ring in sax-3(lf) mutants. To exclude this possibility, we examined the relative position of AMsh glial cells and the nerve ring. Results show that AMsh cell bodies reside posterior to the nerve ring in control animals but are misplaced anterior to the nerve ring in sax-3(lf) animals (Figure 1—figure supplement 1d and e), suggesting that the AMsh migration defect is not a side phenotype coming from a displaced nerve ring. As about 40% of sax-3(lf) animals display defects in head morphogenesis, called ‘vab head’ (Ghenea et al., 2005), it is also possible that AMsh defects could be a result of abnormal head morphology. To rule out this possibility, we examined sax-3(lf) animals without ‘vab head’ phenotypes and found that they displayed the same degree of AMsh defects as in all sax-3(lf) animals, supporting the conclusion that the AMsh migration defects in sax-3(lf) animals are independent of the ‘vab head’ phenotypes (Figure 1c).

The extracellular cleavage fragment of SAX-3/Robo functions as an attractive cue for glial migration

To understand how sax-3 regulates AMsh migration, we carried out tissue-specific rescue experiments. Unexpectedly, as a well-studied receptor, sax-3 does not function cell-autonomously in AMsh migration, and instead expression of sax-3 cDNA in neurons fully rescues sax-3(lf) AMsh phenotypes (Figure 1d, Figure 1—figure supplement 1g). It is even more striking that expression of truncated forms of SAX-3 lacking the intracellular domains (ΔC) or both intracellular and transmembrane domains (Δ[C+TM]) fully rescues sax-3 AMsh defects (Figure 1d, Figure 1—figure supplement 1h), while these transgenes did not affect the sax-3(lf) ‘vab head’ phenotypes (Figure 1—figure supplement 1f and h). A possible explanation for these observations is that a secreted extracellular fragment of SAX-3 may function as an attractive cue released by neurons to guide AMsh migration. To reach this possibility, either sax-3 should have an alternative splicing form that only contains its extracellular region or SAX-3 protein should be cleaved in the extracellular region. The C. elegans genome has been well annotated and studied (Gupta and Sternberg, 2003), and sax-3 does not have any known splicing forms containing only the extracellular region. Interestingly, Drosophila and human Robo have been shown to be cleaved at the FN3 repeats of the extracellular region (Coleman et al., 2010; Seki et al., 2010). As FN3 repeats are highly conserved between C. elegans SAX-3 and Drosophila Robo, we decided to test whether SAX-3 could also be cleaved in the extracellular region. We first generated transgenes expressing SAX-3::HA in the nervous system and collected animals at different developmental stages. As shown in Figure 2a and Figure 2—figure supplement 1a, we were able to detect a cleavage fragment around 70 kDa in different transgene lines and using different anti-HA antibodies. As the HA tag is fused to the C-terminus of SAX-3, based on the molecular weight (about 70 kDa) the cleavage site is likely to be in the FN3 repeats, which is consistent with what was found in Drosophila Robo (Coleman et al., 2010). More importantly, the extracellular cleavage happens from the embryonic 1.5-fold stage to early larval stages, when AMsh glia migrate from their birthplace to the nerve ring region (Figure 2a and Figure 2—figure supplement 1a). As the function of sax-3 in AMsh migration is independent of Slit, we asked whether the cleavage of SAX-3 relied on Slit and found that the cleavage of SAX-3 was not regulated by Slit (Figure 2b and Figure 2—figure supplement 1b). To further confirm that the cleavage site is in the FN3 repeats, we generated transgenes expressing a mini SAX-3 extracellular region that only contains the FN3 repeats, transmembrane domain, and a GFP inserted between the signal peptide and the first FN3 repeat (GFP-FN3-TM) and examined the cleavage products of this fragment. We found that FN3 repeats were sufficient for the cleavage (Figure 2c), and based on the molecular weights of free GFP (27 kDa), GFP-FN3-TM (~80 kDa), and the cleavage product (~45 kDa) we concluded that the cleavage site was in the second repeat of FN3. We then generated transgenes expressing the GFP-FN3-TM fragment lacking one of the three FN3 repeats. As deletion of the first FN3 repeat (FN3-a) caused failure of expression, we were only able to examine the effect of removing the second (FN3-b) or the third FN3 (FN3-c) repeat. We found that the cleavage was abolished if the second FN3 repeat was absent, while deletion of the third FN3 repeat did not affect the cleavage (Figure 2d). Consistent with its important role in SAX-3 cleavage, extracellular cleavage was undetectable in transgenes expressing a truncated form of SAX-3 without the second FN3 repeat (Figure 2e). These data support the conclusion that the cleavage site of SAX-3 is in the second repeat of FN3. To test whether the extracellular cleavage of SAX-3 is essential for its function in AMsh migration, we examined the rescue ability of the truncated form of SAX-3 that lacks the second FN3 repeat and found that this mutant form of SAX-3 completely lost rescue ability (Figure 2f, Figure 2—figure supplement 1c). The extracellular cleavage of SAX-3 appears to show certain specificity for AMsh migration, as the same transgene (SAX-3 ΔFN3-b) fully rescued sax-3(lf) ‘vab head’ phenotypes (Figure 1—figure supplement 1f, Figure 2—figure supplement 1c). To further confirm the role of extracellular SAX-3 cleavage in AMsh migration, we generated a sax-3(yad175) deletion allele that lacks the second repeat of FN3 (FN3-b). sax-3(yad175) decreases sax-3 mRNA to about 50% of that in control animals (Figure 2—figure supplement 1d) and appears to be a weak loss-of-function allele of sax-3, as both sax-3(ky123, presumptive null allele) and sax-3(yad10) show higher penetrance of vab head and AVM guidance defects (Zallen et al., 1999; Figure 1—figure supplement 1f and Figure 2—figure supplement 1e). Despite being a weak allele, sax-3(yad175) exhibits stronger AMsh migration defects when compared with any other sax-3 alleles examined, supporting the critical role of SAX-3 extracellular cleavage in AMsh migration (Figure 2f). To illustrate the function of the extracellular cleavage fragment, we expressed a secreted SAX-3 extracellular fragment containing Ig 1–5 domains and the first FN3 repeat (FN3-a) under pan-neuronal (Punc-33 and Prgef-1) or amphid neuron-specific (Parl-13) promoters, which mimics the cleavage product, and found that this fragment was able to fully rescue sax-3(lf) AMsh defects (Figure 2f, Figure 2—figure supplement 1f). Further experiments show that both Ig domains and the first FN3 repeat (FN3-a) may be important for the function of the SAX-3 extracellular cleavage fragment in AMsh migration (Figure 2f).

Figure 2 with 1 supplement see all

Download asset Open asset

SYG-1/Neph is the receptor for the SAX-3/Robo extracellular cleavage fragment during glial migration

With the discovery of the function of the SAX-3 extracellular cleavage fragment in AMsh glial migration, we hypothesized that the cleavage fragment is released from neurons, where sax-3 is expressed (Zallen et al., 1998), and functions as an attractive cue for glial migration. If that is the case, there should be a receptor in AMsh glia to interact with the SAX-3 cleavage fragment and mediate AMsh migration. To identify the potential receptor for the SAX-3 cleavage fragment, we generated a transgene expressing a functional FLAG::SAX-3 fusion protein, in which a FLAG tag was inserted between the signal peptide and the first Ig domain. We first confirmed that we could detect the developmental-stage-dependent SAX-3 cleavage in this transgene (Figure 3a). Then we carried out immunoprecipitation (IP) using anti-FLAG-coated beads on protein lysate collected from 44 cell and threefold stage embryos and submitted them for proteomics (Figure 3—figure supplement 1a). As the SAX-3 cleavage happens only in threefold but not in 44-cell stage embryos, we focused on membrane proteins that specifically interacted with SAX-3 in threefold stage embryos. Furthermore, Ig superfamily proteins have been shown to be able to interact with each other in many biological processes (Cameron and McAllister, 2018; Wai Wong et al., 2012), so we also focused our analysis on Ig superfamily proteins. syg-1, a nephrin family adhesion molecule (Shen and Bargmann, 2003), caught our attention (Figure 3—figure supplement 1b, Supplementary file 2) because it contains multiple Ig domains in its extracellular regions (Zallen et al., 1998; Shen and Bargmann, 2003). To confirm the interaction between SYG-1 and SAX-3, we first generated a transgene expressing SYG-1::HA in glia and FLAG::SAX-3 in neurons and then examined the interaction between SYG-1::HA and FLAG::SAX-3 by IP using anti-HA-coated beads. As shown in Figure 3b, the extracellular cleavage fragment but not the full-length SAX-3 can bind with SYG-1::HA, suggesting that SYG-1 can directly or indirectly bind with the SAX-3 extracellular cleavage fragment. With these findings, we further examined the function of syg-1 in AMsh migration and found that syg-1 was expressed in AMsh glia during migration and loss-of-function of syg-1(ok3640, null allele) caused similar AMsh migration defects as those in sax-3(lf) mutants (Figure 3c–e, Figure 3—figure supplement 2a). Furthermore, double mutants of syg-1;sax-3 displayed the same extent of defects as in sax-3 single mutants (Figure 3d and e), supporting the conclusion that syg-1 and sax-3 function in the same genetic pathway to regulate AMsh migration. As syg-1 has been shown to function together with syg-2, another Ig super family protein, in synapse formation and axon branching (Shen et al., 2004; Chia et al., 2014), we examined the function of syg-2 in AMsh migration and did not observe any abnormalities in syg-2(lf) mutants (Figure 3e). Consistent with its potential role as a receptor for the SAX-3 cleavage fragment, syg-1 cell autonomously regulates AMsh migration (Figure 3e, Figure 3—figure supplement 2b). Further genetic analysis also showed that the conserved WIRS (WAVE Regulatory receptor sequence) in the SYG-1 C-terminal (Chia et al., 2014) is essential for its function in AMsh migration (Figure 3e), and interruption of WAVE complex function by mutating gex-3, one of the key components of the WAVE complex (Chia et al., 2014), cell autonomously caused similar AMsh migration defects as observed in sax-3(lf) and syg-1(lf) mutants (Figure 3f,g). Finally, to further test the ligand/receptor function of the SAX-3 cleavage fragment and SYG-1, we tested whether AMsh glia can further migrate along the body in transgenic animals that express the cleavage product of SAX-3 in the tail neurons (Pitr-1b) or tail hypodermis (Plin-44) of sax-3(lf) animals (Ou et al., 2010). We found that in these transgenes the migration distance of AMsh was almost three times that of control animals, and loss-of-function in syg-1 completely blocked the over-migration phenotypes in these transgenes (Figure 3h and i). With this evidence we believe that the neuron-released SAX-3 extracellular cleavage fragment functions as an attractive cue, and SYG-1 functions as its receptor to mediate AMsh migration (Figure 4).

Figure 3 with 2 supplements see all

Download asset Open asset

Figure 4

Download asset Open asset

Studies in different organisms show that Robo functions as the receptor for the repulsive guidance cue Slit (Dickson and Gilestro, 2006), and loss-of-function in Robo causes many phenotypes that are shared by Slit mutants (Dickson and Gilestro, 2006). However, genetic studies also showed that some Robo-mutant phenotypes are not related to Slit (Zallen et al., 1998; Hao et al., 2001; Fricke et al., 2001; Hivert et al., 2002; Liu et al., 2004). In C. elegans, there is only one Robo, sax-3, and one Slit, slt-1. sax-3(lf) animals display many phenotypes that are not observed in slt-1 mutants, including embryonic lethality, abnormal head morphology (vab head), and misplacement of neurons (Zallen et al., 1998; Hao et al., 2001). Based on these observations, it has been long speculated that there are Slit-independent Robo regulatory mechanisms. Here we present evidence to show that neuron-expressed Robo/SAX-3 can be cleaved in the extracellular FN3 repeats during development, and the cleaved extracellular fragment can interact with glial-expressed SYG-1/Neph to mediate F-actin organization and to facilitate glial migration.

The extracellular region of Robo is evolutionarily conserved from nematodes to mammals and contains five Ig and three FN3 domains. The extracellular cleavages of Robo appear to be conserved in C. elegans (this study), Drosophila, and human (Coleman et al., 2010; Seki et al., 2010), though different metalloproteases were utilized in Drosophila and human. Recent biochemical and structural studies show that the extracellular domains of Robo can dimerize through the Ig domains, and the dimerization may be inhibited by the FN3 domain in the absence of Slit (Hivert et al., 2002; Liu et al., 2004; Zakrys et al., 2014; Aleksandrova et al., 2018; Barak et al., 2019; Yamamoto et al., 2019; Pak et al., 2020). The cleavages of the extracellular domains provide possibility for additional regulatory mechanisms of Robo. Since the extracellular SAX-3/Robo cleavage fragment lacks the inhibitory second FN3 domain, it will be expected to strongly bind with itself or with the full length SAX-3/Robo, and these interactions may play a regulatory role in the activation of Robo downstream signals. In this study we show that the SAX-3/Robo extracellular cleavage fragment can bind with the Ig domain-containing adhesion molecule SYG-1/Neph to regulate the WAVE complex. Since sax-3/Robo and syg-1/ Neph are both expressed in and functionally important for neurons and other cells, it is possible that interactions between the SAX-3 cleaved extracellular fragment and syg-1/Neph also play functions beyond glial migration. The potential dimerization of SAX-3/Robo extracellular cleavage fragments may also mediate crosstalk between syg-1/Neph and sax-3/Robo signals. The extracellular cleavage of SAX-3/Robo generated two fragments: one has all the Ig and the first FN3 domains while the other contains the rest of the FN3, transmembrane, and the intracellular domains. While the released SAX-3/Robo extracellular cleavage fragment functions as a ligand for SYG-1/Neph and has potential to interact with the full length SAX-3/Robo, the membrane-anchored fragment may also play an active role in neuronal development.

To achieve their vital roles in the nervous system and to form functional units with neurons, glia need to migrate over what are often long distances from their birthplace to the appropriate regions (Gilmour et al., 2002; Jarjour and Kennedy, 2004; Kinrade et al., 2001; Klämbt, 2009; Barres, 2008; Fields et al., 2015; Mori et al., 2005). The migration of glia relies on guidance cues and shares many similar mechanisms with neurons (Kinrade et al., 2001; Jarjour et al., 2003; Sasse and Klämbt, 2016; Spassky et al., 2002; Unni et al., 2012). Our studies uncover a surprising role of Robo receptors – an attractive cue for glial migration – and identify SYG-1/Neph as the glial receptor for the neuronal-released Robo cleavage fragment, suggesting that the function and regulation of guidance molecules are more complicated than anticipated.

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Dong Yan (dong.yan@duke.edu).

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Aleksandrova N
   2. Gutsche I
   3. Kandiah E
   4. Avilov SV
   5. Petoukhov MV
   6. Seiradake E
   7. McCarthy AA

   (2018) Robo1 forms a compact Dimer-of-Dimers assembly

   Structure 26:320–328.

   https://doi.org/10.1016/j.str.2017.12.003

   • PubMed
   • Google Scholar
2. 1. Bacaj T
   2. Tevlin M
   3. Lu Y
   4. Shaham S

   (2008) Glia are essential for sensory organ function in C. elegans

   Science 322:744–747.

   https://doi.org/10.1126/science.1163074

   • PubMed
   • Google Scholar
3. 1. Barak R
   2. Yom-Tov G
   3. Guez-Haddad J
   4. Gasri-Plotnitsky L
   5. Maimon R
   6. Cohen-Berkman M
   7. McCarthy AA
   8. Perlson E
   9. Henis-Korenblit S
   10. Isupov MN
   11. Opatowsky Y

   (2019) Structural principles in robo activation and Auto-inhibition

   Cell 177:272–285.

   https://doi.org/10.1016/j.cell.2019.02.004

   • PubMed
   • Google Scholar
4. 1. Barres BA

   (2008) The mystery and magic of Glia: a perspective on their roles in health and disease

   Neuron 60:430–440.

   https://doi.org/10.1016/j.neuron.2008.10.013

   • PubMed
   • Google Scholar
5. 1. Branchfield K
   2. Nantie L
   3. Verheyden JM
   4. Sui P
   5. Wienhold MD
   6. Sun X

   (2016) Pulmonary neuroendocrine cells function as airway sensors to control lung immune response

   Science 351:707–710.

   https://doi.org/10.1126/science.aad7969

   • PubMed
   • Google Scholar
6. 1. Brennaman LH
   2. Moss ML
   3. Maness PF

   (2014) EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease

   Journal of Neurochemistry 128:267–279.

   https://doi.org/10.1111/jnc.12468

   • PubMed
   • Google Scholar
7. 1. Brenner S

   (1974)

   The genetics of Caenorhabditis elegans

   Genetics 77:71–94.

   • PubMed
   • Google Scholar
8. 1. Cameron S
   2. McAllister AK

   (2018) Immunoglobulin-Like receptors and their impact on wiring of brain synapses

   Annual Review of Genetics 52:567–590.

   https://doi.org/10.1146/annurev-genet-120417-031513

   • PubMed
   • Google Scholar
9. 1. Chia PH
   2. Chen B
   3. Li P
   4. Rosen MK
   5. Shen K

   (2014) Local F-actin network links synapse formation and axon branching

   Cell 156:208–220.

   https://doi.org/10.1016/j.cell.2013.12.009

   • PubMed
   • Google Scholar
10. 1. Coleman HA
    2. Labrador JP
    3. Chance RK
    4. Bashaw GJ

    (2010) The adam family metalloprotease kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline

    Development 137:2417–2426.

    https://doi.org/10.1242/dev.047993

    • PubMed
    • Google Scholar
11. 1. Dickson BJ
    2. Gilestro GF

    (2006) Regulation of commissural axon pathfinding by slit and its robo receptors

    Annual Review of Cell and Developmental Biology 22:651–675.

    https://doi.org/10.1146/annurev.cellbio.21.090704.151234

    • PubMed
    • Google Scholar
12. 1. Fields RD
    2. Woo DH
    3. Basser PJ

    (2015) Glial Regulation of the Neuronal Connectome through Local and Long-Distant Communication

    Neuron 86:374–386.

    https://doi.org/10.1016/j.neuron.2015.01.014

    • Google Scholar
13. 1. Fricke C
    2. Lee JS
    3. Geiger-Rudolph S
    4. Bonhoeffer F
    5. Chien CB

    (2001) Astray, a zebrafish roundabout homolog required for retinal axon guidance

    Science 292:507–510.

    https://doi.org/10.1126/science.1059496

    • PubMed
    • Google Scholar
14. 1. Ghenea S
    2. Boudreau JR
    3. Lague NP
    4. Chin-Sang ID

    (2005) The VAB-1 eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis

    Development 132:3679–3690.

    https://doi.org/10.1242/dev.01947

    • PubMed
    • Google Scholar
15. 1. Gilmour DT
    2. Maischein HM
    3. Nüsslein-Volhard C

    (2002) Migration and function of a glial subtype in the vertebrate peripheral nervous system

    Neuron 34:577–588.

    https://doi.org/10.1016/S0896-6273(02)00683-9

    • PubMed
    • Google Scholar
16. 1. Grieshammer U
    2. Ma L
    3. Plump AS
    4. Wang F
    5. Tessier-Lavigne M
    6. Martin GR

    (2004) SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site

    Developmental Cell 6:709–717.

    https://doi.org/10.1016/S1534-5807(04)00108-X

    • PubMed
    • Google Scholar
17. 1. Gupta BP
    2. Sternberg PW

    (2003) The draft genome sequence of the nematode Caenorhabditis briggsae

    Genome Biology 4:238.

    https://doi.org/10.1186/gb-2003-4-12-238

    • Google Scholar
18. 1. Hao JC
    2. Yu TW
    3. Fujisawa K
    4. Culotti JG
    5. Gengyo-Ando K
    6. Mitani S
    7. Moulder G
    8. Barstead R
    9. Tessier-Lavigne M
    10. Bargmann CI

    (2001) C. elegans slit acts in Midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor

    Neuron 32:25–38.

    https://doi.org/10.1016/S0896-6273(01)00448-2

    • PubMed
    • Google Scholar
19. 1. Heiman MG
    2. Shaham S

    (2009) DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration

    Cell 137:344–355.

    https://doi.org/10.1016/j.cell.2009.01.057

    • PubMed
    • Google Scholar
20. 1. Hinck L

    (2004) The versatile roles of "axon guidance" cues in tissue morphogenesis

    Developmental Cell 7:783–793.

    https://doi.org/10.1016/j.devcel.2004.11.002

    • PubMed
    • Google Scholar
21. 1. Hivert B
    2. Liu Z
    3. Chuang CY
    4. Doherty P
    5. Sundaresan V

    (2002) Robo1 and Robo2 are homophilic binding molecules that promote axonal growth

    Molecular and Cellular Neuroscience 21:534–545.

    https://doi.org/10.1006/mcne.2002.1193

    • PubMed
    • Google Scholar
22. 1. Jarjour AA
    2. Manitt C
    3. Moore SW
    4. Thompson KM
    5. Yuh SJ
    6. Kennedy TE

    (2003) Netrin-1 is a chemorepellent for oligodendrocyte precursor cells in the embryonic spinal cord

    The Journal of Neuroscience 23:3735–3744.

    https://doi.org/10.1523/JNEUROSCI.23-09-03735.2003

    • PubMed
    • Google Scholar
23. 1. Jarjour AA
    2. Kennedy TE

    (2004) Oligodendrocyte precursors on the move: mechanisms directing migration

    The Neuroscientist 10:99–105.

    https://doi.org/10.1177/1073858403260751

    • PubMed
    • Google Scholar
24. 1. Killeen MT
    2. Sybingco SS

    (2008) Netrin, slit and wnt receptors allow axons to choose the Axis of migration

    Developmental Biology 323:143–151.

    https://doi.org/10.1016/j.ydbio.2008.08.027

    • PubMed
    • Google Scholar
25. 1. Kinrade EF
    2. Brates T
    3. Tear G
    4. Hidalgo A

    (2001)

    Roundabout signalling, cell contact and trophic support confine longitudinal Glia and axons in the Drosophila CNS

    Development 128:207–216.

    • PubMed
    • Google Scholar
26. 1. Klämbt C

    (2009) Modes and regulation of glial migration in vertebrates and invertebrates

    Nature Reviews Neuroscience 10:769–779.

    https://doi.org/10.1038/nrn2720

    • PubMed
    • Google Scholar
27. 1. Liu Z
    2. Patel K
    3. Schmidt H
    4. Andrews W
    5. Pini A
    6. Sundaresan V

    (2004) Extracellular ig domains 1 and 2 of robo are important for ligand (Slit) binding

    Molecular and Cellular Neuroscience 26:232–240.

    https://doi.org/10.1016/j.mcn.2004.01.002

    • PubMed
    • Google Scholar
28. 1. Mommersteeg MT
    2. Andrews WD
    3. Ypsilanti AR
    4. Zelina P
    5. Yeh ML
    6. Norden J
    7. Kispert A
    8. Chédotal A
    9. Christoffels VM
    10. Parnavelas JG

    (2013) Slit-roundabout signaling regulates the development of the cardiac systemic venous return and pericardium

    Circulation Research 112:465–475.

    https://doi.org/10.1161/CIRCRESAHA.112.277426

    • PubMed
    • Google Scholar
29. 1. Mori T
    2. Buffo A
    3. Götz M

    (2005) The novel roles of glial cells revisited: the contribution of radial Glia and astrocytes to neurogenesis

    Current Topics in Developmental Biology 69:67–99.

    https://doi.org/10.1016/S0070-2153(05)69004-7

    • PubMed
    • Google Scholar
30. 1. Neuhaus-Follini A
    2. Bashaw GJ

    (2015) The intracellular domain of the frazzled/DCC receptor is a transcription factor required for commissural axon guidance

    Neuron 87:751–763.

    https://doi.org/10.1016/j.neuron.2015.08.006

    • PubMed
    • Google Scholar
31. 1. Oikonomou G
    2. Shaham S

    (2011) The Glia of Caenorhabditis elegans

    Glia 59:1253–1263.

    https://doi.org/10.1002/glia.21084

    • PubMed
    • Google Scholar
32. 1. Ou CY
    2. Poon VY
    3. Maeder CI
    4. Watanabe S
    5. Lehrman EK
    6. Fu AK
    7. Park M
    8. Fu WY
    9. Jorgensen EM
    10. Ip NY
    11. Shen K

    (2010) Two Cyclin-Dependent kinase pathways are essential for polarized trafficking of presynaptic components

    Cell 141:846–858.

    https://doi.org/10.1016/j.cell.2010.04.011

    • PubMed
    • Google Scholar
33. 1. Packer JS
    2. Zhu Q
    3. Huynh C
    4. Sivaramakrishnan P
    5. Preston E
    6. Dueck H
    7. Stefanik D
    8. Tan K
    9. Trapnell C
    10. Kim J
    11. Waterston RH
    12. Murray JI

    (2019) A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution

    Science 365:eaax1971.

    https://doi.org/10.1126/science.aax1971

    • PubMed
    • Google Scholar
34. 1. Pak JS
    2. DeLoughery ZJ
    3. Wang J
    4. Acharya N
    5. Park Y
    6. Jaworski A
    7. Özkan E

    (2020) NELL2-Robo3 complex structure reveals mechanisms of receptor activation for axon guidance

    Nature Communications 11:1489.

    https://doi.org/10.1038/s41467-020-15211-1

    • PubMed
    • Google Scholar
35. 1. Perkins LA
    2. Hedgecock EM
    3. Thomson JN
    4. Culotti JG

    (1986) Mutant sensory cilia in the nematode Caenorhabditis elegans

    Developmental Biology 117:456–487.

    https://doi.org/10.1016/0012-1606(86)90314-3

    • PubMed
    • Google Scholar
36. 1. Sasakura H
    2. Inada H
    3. Kuhara A
    4. Fusaoka E
    5. Takemoto D
    6. Takeuchi K
    7. Mori I

    (2005) Maintenance of neuronal positions in organized ganglia by SAX-7, a Caenorhabditis elegans homologue of L1

    The EMBO Journal 24:1477–1488.

    https://doi.org/10.1038/sj.emboj.7600621

    • Google Scholar
37. 1. Sasse S
    2. Klämbt C

    (2016) Repulsive epithelial cues direct glial migration along the nerve

    Developmental Cell 39:696–707.

    https://doi.org/10.1016/j.devcel.2016.11.016

    • PubMed
    • Google Scholar
38. 1. Seeger M
    2. Tear G
    3. Ferres-Marco D
    4. Goodman CS

    (1993) Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline

    Neuron 10:409–426.

    https://doi.org/10.1016/0896-6273(93)90330-T

    • PubMed
    • Google Scholar
39. 1. Seki M
    2. Watanabe A
    3. Enomoto S
    4. Kawamura T
    5. Ito H
    6. Kodama T
    7. Hamakubo T
    8. Aburatani H

    (2010) Human ROBO1 is cleaved by metalloproteinases and gamma-secretase and migrates to the nucleus in Cancer cells

    FEBS Letters 584:2909–2915.

    https://doi.org/10.1016/j.febslet.2010.05.009

    • PubMed
    • Google Scholar
40. 1. Shaham S

    (2010) Chemosensory organs as models of neuronal synapses

    Nature Reviews Neuroscience 11:212–217.

    https://doi.org/10.1038/nrn2740

    • PubMed
    • Google Scholar
41. 1. Shaham S

    (2015) Glial Development and Function in the Nervous System of Caenorhabditis elegans

    Cold Spring Harbor Perspectives in Biology 7:a020578.

    https://doi.org/10.1101/cshperspect.a020578

    • Google Scholar
42. 1. Shen K
    2. Fetter RD
    3. Bargmann CI

    (2004) Synaptic specificity is generated by the synaptic guidepost protein SYG-2 and its receptor, SYG-1

    Cell 116:869–881.

    https://doi.org/10.1016/S0092-8674(04)00251-X

    • PubMed
    • Google Scholar
43. 1. Shen K
    2. Bargmann CI

    (2003) The immunoglobulin superfamily protein SYG-1 determines the location of specific synapses in C. elegans

    Cell 112:619–630.

    https://doi.org/10.1016/S0092-8674(03)00113-2

    • PubMed
    • Google Scholar
44. 1. Singhvi A
    2. Liu B
    3. Friedman CJ
    4. Fong J
    5. Lu Y
    6. Huang X-Y
    7. Shaham S

    (2016) A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC

    Cell 165:936–948.

    https://doi.org/10.1016/j.cell.2016.03.026

    • Google Scholar
45. 1. Spassky N
    2. de Castro F
    3. Le Bras B
    4. Heydon K
    5. Quéraud-LeSaux F
    6. Bloch-Gallego E
    7. Chédotal A
    8. Zalc B
    9. Thomas JL

    (2002) Directional guidance of oligodendroglial migration by class 3 semaphorins and netrin-1

    The Journal of Neuroscience 22:5992–6004.

    https://doi.org/10.1523/JNEUROSCI.22-14-05992.2002

    • PubMed
    • Google Scholar
46. 1. Unni DK
    2. Piper M
    3. Moldrich RX
    4. Gobius I
    5. Liu S
    6. Fothergill T
    7. Donahoo A-LS
    8. Baisden JM
    9. Cooper HM
    10. Richards LJ

    (2012) Multiple slits regulate the development of midline glial populations and the corpus callosum

    Developmental Biology 365:36–49.

    https://doi.org/10.1016/j.ydbio.2012.02.004

    • Google Scholar
47. 1. Viveiros R
    2. Hutter H
    3. Moerman DG

    (2011) Membrane extensions are associated with proper anterior migration of muscle cells during Caenorhabditis elegans embryogenesis

    Developmental Biology 358:189–200.

    https://doi.org/10.1016/j.ydbio.2011.07.026

    • PubMed
    • Google Scholar
48. 1. Wai Wong C
    2. Dye DE
    3. Coombe DR

    (2012) The role of immunoglobulin superfamily cell adhesion molecules in Cancer metastasis

    International Journal of Cell Biology 2012:1–9.

    https://doi.org/10.1155/2012/340296

    • Google Scholar
49. 1. Wang LJ
    2. Zhao Y
    3. Han B
    4. Ma YG
    5. Zhang J
    6. Yang DM
    7. Mao JW
    8. Tang FT
    9. Li WD
    10. Yang Y
    11. Wang R
    12. Geng JG

    (2008) Targeting Slit-Roundabout signaling inhibits tumor angiogenesis in chemical-induced squamous cell carcinogenesis

    Cancer Science 99:510–517.

    https://doi.org/10.1111/j.1349-7006.2007.00721.x

    • PubMed
    • Google Scholar
50. 1. Ward S
    2. Thomson N
    3. White JG
    4. Brenner S

    (1975) Electron microscopical reconstruction of the anterior sensory anatomy of the nematode Caenorhabditis elegans.?2uu

    The Journal of Comparative Neurology 160:313–337.

    https://doi.org/10.1002/cne.901600305

    • PubMed
    • Google Scholar
51. 1. Wu JY
    2. Feng L
    3. Park HT
    4. Havlioglu N
    5. Wen L
    6. Tang H
    7. Bacon KB
    8. Jiang ZH
    9. Zhang XC
    10. Rao Y

    (2001) The neuronal repellent slit inhibits leukocyte chemotaxis induced by chemotactic factors

    Nature 410:948–952.

    https://doi.org/10.1038/35073616

    • PubMed
    • Google Scholar
52. 1. Yamamoto N
    2. Kashiwagi M
    3. Ishihara M
    4. Kojima T
    5. Maturana AD
    6. Kuroda S
    7. Niimi T

    (2019) Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2

    Journal of Biological Chemistry 294:4693–4703.

    https://doi.org/10.1074/jbc.RA118.005819

    • PubMed
    • Google Scholar
53. 1. Zakrys L
    2. Ward RJ
    3. Pediani JD
    4. Godin AG
    5. Graham GJ
    6. Milligan G

    (2014) Roundabout 1 exists predominantly as a basal dimeric complex and this is unaffected by binding of the ligand Slit2

    Biochemical Journal 461:61–73.

    https://doi.org/10.1042/BJ20140190

    • PubMed
    • Google Scholar
54. 1. Zallen JA
    2. Yi BA
    3. Bargmann CI

    (1998) The conserved immunoglobulin superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C. elegans

    Cell 92:217–227.

    https://doi.org/10.1016/S0092-8674(00)80916-2

    • PubMed
    • Google Scholar
55. 1. Zallen JA
    2. Kirch SA
    3. Bargmann CI

    (1999)

    Genes required for axon pathfinding and extension in the C-elegans nerve ring

    Development 126:3679–3692.

    • PubMed
    • Google Scholar
56. 1. Zhang A
    2. Noma K
    3. Yan D

    (2020a) Regulation of gliogenesis by lin-32/Atoh1 in Caenorhabditis elegans

    G3: Genes, Genomes, Genetics 10:401547.

    https://doi.org/10.1534/g3.120.401547

    • Google Scholar
57. 1. Zhang A
    2. Ackley BD
    3. Yan D

    (2020b) Vitamin B12 regulates glial migration and synapse formation through Isoform-Specific control of PTP-3/LAR PRTP expression

    Cell Reports 30:3981–3988.

    https://doi.org/10.1016/j.celrep.2020.02.113

    • PubMed
    • Google Scholar

Author details

1. Zhongwei Qu

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6994-0530

2. Albert Zhang

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1310-5680

3. Dong Yan

   1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States
   2. Department of Neurobiology, Regeneration Next Initiative, Duke Center for Neurodegeneration and Neurotherapeutics, and Duke Institute for Brain Sciences, Duke University Medical Center, Durham, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review and editing

   For correspondence

   dong.yan@duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7542-1251

• 991

  views

• 131

  downloads

• 4

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.