RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin. Author Summary Neural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.


Introduction
The connectivity of neuronal circuits is established through properly guided 47 axons which form functional synaptic connections. The growing axon is guided to its 48 target by the motile, actin-based growth cone at the tip of the growing neurite. Growth 49 cone response to extracellular guidance cues allows the axon to extend, retract, turn accumulation. In sum, results reported here show that RHGF-1 and RHO-1 are key 115 inhibitors of growth cone protrusion and MT + end accumulation and act with UNC-5 in 116 protrusion, but not growth cone polarity. compared to wild-type VD growth cones ( Figure 1A-B, E). Growth cone area was 131 increased, but not significantly so. These results indicate that RHO-1 activity inhibits 132 growth cone protrusion. 133 We used a transgenic RNAi approach to knock down rho-1 in the VD/DD motor RHO-1 is required to limit EBP-2::GFP puncta accumulation in VD growth cones. 148 Previous studies indicate that in VD growth cones, F-actin accumulates at the 149 dorsal, protrusive edge of the growth cone and acts as a polarity mark to specify 150 protrusion in this region (Figure 2A   Constitutively-active rho-1(G14V) resulted in fewer EBP-2::GFP puncta, 165 consistent with reduced growth cone protrusion ( Figure 2E). F-actin polarity was also 166 abolished, with distribution along the periphery of the entire growth cone (Figure 2A    rhgf-1(gk292502) produces a premature stop just before the C1 domain ( Figure 3A).

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rhgf-1 mutants each displayed increased growth cone area and longer filopodial 11 185 protrusions compared to wild-type ( Figure 3B-F). The dorsally-biased polarity of growth 186 cone protrusion was not significantly affected by rhgf-1 mutation ( Figure 3G-I). These 187 data indicate that RHGF-1 is normally required to limit the extent of growth cone 188 protrusion, but does not regulate growth cone polarity, similar to rho-1. 189 The Drosophila RHGF-1 homolog DRhoGEF2 is a key regulator of 4D-F). These results indicate that RHGF-1 might act with RHO-1 to inhibit growth cone 196 protrusion by excluding MT+ ends from entering the growth cone periphery.

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The results above indicate that the VD growth cones of activated rho-1(G14V) 198 displayed reduced protrusion, and that those of rhgf-1 loss of function were overly-

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Our results here show that the small GTPases RHO-1 and the Rho Guanine 279 nucleotide Exchange factor RHGF-1 mediate inhibition of growth cone protrusion and 280 are required to limit MT+ -end accumulation in growth cones, similar to UNC-5 and 281 UNC-33. However, RHO-1 and RHGF-1 had no effect on growth cone polarity (i.e. 282 mutants did not affect dorsally-biased distribution of filopodial protrusion and F-actin).  (Figures 1 and 2). Notably, neither dominant-306 negative RHO-1(T19N) or rho-1(RNAi) resulted in altered growth cone polarity and F-307 actin dorsal bias (Figures 1 and 2), suggesting that RHO-1 might specifically affect 308 growth cone protrusion but not polarity.  Loss of rhgf-1 resulted in increased growth cone protrusion and accumulation of 332 EBP-2::GFP, similar to but more pronounced than dominant-negative RHO-1(T19N) 333 and rho-1(RNAi) (Figures 3 and 4). Furthermore, rhgf-1 mutants had no effect on growth 334 cone polarity of protrusion or F-actin distribution (Figure 4). RHGF-1 might be an    Interestingly, despite their small size, inhibited unc-33; rho-1(G14V) growth 388 cones displayed increased EBP-2 puncta compared to wild-type animals, but 389 significantly lower than unc-33 mutants alone ( Figure 12). Thus, activated RHO-390 1(G14V) can fully suppress excess protrusion, but not EBP-2::GFP accumulation, of 391 unc-33 mutants. Together, these results suggest that UNC-33 is required for activated 392 RHO-1(G14V) to restrict MTs from growth cones. They also suggest that RHO-1 has a 393 role in protrusion that is independent of MT accumulation, as protrusion was reduced in      were defined, and the EBP-2::GFP puncta in the growth cone were counted. For each 499 genotype, the puncta number for many growth cones (≥25 unless otherwise noted) was 500 determined. Puncta number displayed high variability within and between genotypes, so 501 box-and-whiskers plots (Graphpad Prism) were used to accurately depict this variation.

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The grey boxes represent the upper and lower quartiles of the data set, and the 503 "whiskers" represent the high and low values. Dots represent major outliers.

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Significance of difference was determined by a two-sided t-test with unequal variance.

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Transgenic RNA-mediated gene interference (RNAi) 506 We used a cell-specific transgenic RNAi approach as described previously (ESPOSITO et  The authors thank the members of the Lundquist and Ackley labs for discussion and E. 517 Struckhoff for technical assistance. Some strains were provided by the CGC, which is 518 funded by NIH Office of Research Infrastructure Programs (P40 OD010440).