Gujar, Mahekta et al. (2015) International Worm Meeting "UNC-6/Netrin Repulsive Axon Guidance Involves UNC-33/CRMP and Flavin Monooxygenases."

Gujar, Mahekta, Lundquist, Erik

[International Worm Meeting, 2015]

Developing axons are led by growth cones, which are dynamic actin-based structures that sense and respond to extracellular guidance cues, driving the forward motion of the axon towards the target of migration. The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5/UNC-40 heterodimer, inhibits growth cone protrusion (Norris et al., 2011). Expression of an activated MYR::UNC-40 construct in the repelled VD axons constitutively inhibits protrusion. We used MYR::UNC-40 to delineate a pathway involving UNC-73/Trio, the Rac GTPases CED-10 and MIG-2, and the microtubule-interacting protein UNC-33/CRMP that inhibits growth cone protrusion downstream of MYR::UNC-40 (Norris et al., 2014). While CRMPs have been implicated in semaphorin-mediated collapse, our data suggest that they also control UNC-6/Netrin-mediated repulsion.In unc-33 mutants, excess microtubule plus ends were observed in growth cones, correlating with excess growth cone protrusion. The MICAL proteins are flavin monooxygenases that cause actin depolymerization involved in growth cone collapse downstream of semaphorin (Hung et al., 2010). The C. elegans genome does not encode a single MICAL-like gene. However, it encodes five flavin monooxygenases (fmo-1-5) (Petalcorin et al., 2005), similar to that found in MICAL, as well as an actin-interacting protein, ehbp-1 (Shi et al., 2010), which is similar to the non-FMO portion of MICAL. Here we show that the ehbp-1 and fmo genes may play a role in Netrin directed repulsive guidance mediated through UNC-40/DCC and UNC-5. A subset of the fmo genes and ehbp-1 showed VD/DD axon guidance and branching defects and enhanced unc-40 VD/DD guidance defects. When placed in a MYR::UNC-40 background these mutants partially suppressed myr::unc-40. Further studies will be directed at determining if these genes act downstream of the Rac GTPases and UNC-73/Trio, if they are expressed in and act cell-autonomously in neurons to regulate axon guidance, and if they affect the actin cytoskeleton of the growth cone.In sum, we have defined a novel pathway that inhibits growth cone protrusion downstream of UNC-6/Netrin. While CRMP has been shown to mediate semaphorin induced growth cone collapse, our results implicate UNC-33/CRMP in UNC-6/Netrin repulsive axon guidance and that that the MICAL-like fmo genes and ehbp-1 also act in inhibition, possibly via the actin cytoskeleton.

Gujar, Mahekta et al. (2017) International Worm Meeting "RHO-1 and the activator RHGF-1/RhoGEF are required for EBP-2 distribution in growth cones."

Stricker, Aubrie, Lundquist, Erik, Gujar, Mahekta

[International Worm Meeting, 2017]

Previous studies showed that UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5 control axon guidance by regulating protrusion of growth cone lamellipodia and filopodia (Norris et al., 2011; Norris et al., 2014). UNC-6 stimulates protrusion in growth cones attracted to UNC-6 via the UNC-40 homodimeric receptor, and inhibits protrusion in growth cones repelled from UNC-6 via the UNC-5/UNC-40 heterodimeric receptor. Protrusion via UNC-40 requires CDC-42, the Rac GTPases MIG-2 and CED-10, the Rac GEF TIAM-1, and the actin modulating molecules Arp2/3 and UNC-115/abLIM. This results in polarized distribution to the leading edge of the growth cone. We delineated a pathway downstream of UNC-5/UNC-40 required to inhibit protrusion that also involves the Rac GTPases and a distinct GEF, UNC-73/Trio, as well as the microtubule-interacting protein UNC-33, similar to Collapsin response mediating protein (CRMP). We used EBP-2::GFP expression in VD neurons to monitor MT + ends in VD growth cones. We found that mutations in components of the UNC-5, UNC-33/CRMP pathway result in excess EBP-2::GFP puncta in VD growth cones, suggesting that this pathway might normally restrict MT + end entry into the growth cones to inhibit growth cone protrusion. Interestingly, a mutation affecting specifically the Rac GEF domain of unc-73 did not affect EBP-2::GFP distribution in growth cones despite having excessively protrusive growth cones. F-actin distribution was affected, indicating that inhibition of protrusion can act through both actin and MTs. We find that the MT-dependent mechanism involves RHO-1 and its upstream activator RHGF-1/PDZ RhoGEF. rhgf-1 mutants show large protrusive growth cones similar to those seen in unc-5 and unc-33 mutants and displayed greatly increased numbers of EBP-2::GFP puncta in VD growth cones and protrusions. The dominant negative form of RHO-1, rho-1(T19N) also resembles unc-5, with excess lamellipodial and filopodial protrusions and show increased EBP-2::GFP puncta. In sum, these studies indicate that growth cone protrusion can be inhibited by UNC-6/Netrin signaling by restricting MT + ends from growth cones, and that ectopic MT + ends in growth cones drive ectopic protrusion.

Josephson, Matthew P et al. (2013) International Worm Meeting "Transcriptome analysis reveals genes regulated by MAB-5/Hox in posterior migration of Q neuroblast descendants."

Macdonald, Stuart, Lundquist, Erik, Tamayo, Joel, Josephson, Matthew P, Gujar, Mahekta

[International Worm Meeting, 2013]

The Q neuroblasts QR and QL are born in the same location along the longitudinal axis then migrate in opposite directions exposing QL but not QR to an EGL-20/Wnt signal. This Wnt signal induces expression of the homeobox transcription factor MAB-5/Hox, a determinant for posterior migration, causing the QL descendants (SDQL, PVM, and PQR) to migrate posteriorly. mab-5 loss of function (lof) results in anterior migration of QL descendants, and mab-5(e1751), a gain of function (gof) allele that expresses MAB-5 in both QL and QR, results in posterior migration of QR descendants. To identify genes regulated by MAB-5 that drive posterior migration, RNA seq was conducted on wild-type, mab-5lof, and mab-5gof. Genes were found that were upregulated or downregulated in lof or gof backgrounds. Gene ontology term analysis showed that these were significantly enriched for genes encoding secreted and transmembrane molecules that mediate interactions with the extracellular matrix. We used RNAi to perturb genes that were upregulated in mab-5gof and downregulated in mab-5lof, with the idea that these might be genes that are upregulated by MAB-5 to drive posterior migration. We found that RNAi of approximately a third of these genes (17) modified posterior QR descendant AQR migration in mab-5gof, indicating that they are required by MAB-5 to drive posterior migration. Existing mutations within genes were used to confirm RNAi results. The MAP kinase activated protein kinase mak-1, and the C. elegans F-spondin homolog spon-1 were both upregulated in mab-5gof and both suppressed the mab-5gof posterior AQR migration. Q cell specific RNAi experiments on these genes did not result in suppression, suggesting a non cell-autonomous role in posterior migration. A ChiP-seq study previously identified mak-1 but not spon-1 as a direct target of MAB-5 (Niu et al, 2011). The data taken together suggest that mak-1 is a direct target of MAB-5 required for posterior migration of Q descendants, and spon-1 may be indirectly controlled by MAB-5 in posterior migration.

Ferguson, Kelsey et al. (2021) International Worm Meeting "Membrane-anchored UNC-6/Netrin reveals roles of both close- and long-range interactions in regulating VD growth cone dorsal outgrowth"

Mahadik, Snehal, Ferguson, Kelsey, Lundquist, Erik

[International Worm Meeting, 2021]

The UNC-6/Netrin guidance cue directs dorsal-ventral axon pathfinding and is expressed in ventral cord motor neurons. Classically, UNC-6 was thought to form a ventral-to-dorsal gradient that was interpreted dynamically by growth cones. Our previous VD growth cone imaging studies indicated that the mechanism is likely more complex, involving discrete aspects of growth cone polarity coupled with regulation of growth cone protrusion. This polarity/protrusion model parts from the classical gradient model in important ways. First, growth cone polarity is separable from growth cone protrusion (Gujar et al., 2019). Second, UNC-40/DCC and UNC-5 receptors both have roles in growth cones that grow dorsally away from UNC-6 (Norris and Lundquist, 2011; Norris et al., 2014). To further test the polarity/protrusion model, we constructed a membrane-anchored UNC-6 molecule by adding a C-terminal transmembrane domain through genome editing called unc-6(lq154). This mutant is predicted to lack diffusible UNC-6. The AVM neuron is born laterally and extends an axon ventrally toward UNC-6. As there is no contact between AVM and UNC-6-expressing cells, diffusible UNC-6 is predicted to be required to guide the AVM axon. unc-6(lq154) animals display axon guidance defects to the same extent as unc-6(ev400) null mutants, consistent with the idea that no diffusible UNC-6 is produced. In contrast, VD/DD axon guidance defects of unc-6(lq154) were less severe than those of unc-6(ev400), suggesting that unc-6(lq154) retains some function in VD/DD axon guidance. unc-6(ev400) VD growth cones were unpolarized, whereas polarity in VD growth cones near the ventral surface in unc-6(lq154) was normal. Growth cones further from the ventral surface, in the dorsal half of the animal, were unpolarized in unc-6(lq154). This result suggests that initial growth cone polarity is normal in unc-6(lq154), but polarity is lost as the growth cones migrate dorsally. Possibly, a close-range or contact-mediated interaction of UNC-6 and UNC-5 polarizes the growth cone, but longer-range diffusible UNC-6 is required to maintain this polarity. Preliminary studies indicate that hypomorphic unc-5 alleles, which are predicted to affect only the long isoforms of UNC-5, show a similar phenotype. This leads to the notion that the short unc-5B isoform mediates close-range polarity, possibly a contact-mediated event, whereas the long isoforms might mediate maintenance of polarity over longer distances requiring diffusible UNC-6.