Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

Abstract

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

Keywords: Exo84; Exocyst; LET-60; LIN-45; RAL-1; RAP-1; RGL-1; RalBP1; Sec5.

Figure 1:. Disruption of RGL-1/RalGEF enhances mutant defects in CAN migration.

A) A gene model of rgl-1 with protein domains indicated above the model and genetic tools indicated below. These include a putative GEF-dead missense allele, gk275304 (R361Q), nonsense allele gk275305 (W163*), in-frame deletion ok1921, and out-of-frame deletion tm2255. The yellow-colored exon encodes 20 residues that are not conserved in other species and is present in ~5% of RNAseq reads. B, C) Epifluorescent photomicrographs of CAN position at the L1 stage. L1 CANs are positioned just anterior to the gonad primordium, HSNs just posterior. CANs and HSNs, indicated by arrows are bilaterally symmetric, but cells on the other side are out of the plane of focus in these images. Positioning of CANs and HSNs was visualized using the otIs33 GFP reporter in B) wild-type and C) mig-2(gm38gf) backgrounds. Scale bar is 10 μm. D-L) CAN positioning was recorded as decile scores from 0 to 11. 10 indicates the wild-type position, 11 indicates over-migration, and 0 indicates a missing CAN, presumed to have not exited the head but not discernable amongst other GFP-labelled neurons. Any two strains represented by a graph were scored at the same time and were grown on the same OP50 lawn at the same temperature. Positioning of CANs were scored in the otIs33[Pkal-1::GFP] background unless otherwise noted. D) mig-2(gm38gf) confers a moderate migration defect. E, F) The rgl-1 deletions tm2255 and ok1921, respectively, enhanced the migration defect of gm38gf. G, H) The rgl-1 nonsense and GEF-specific alleles, gk275305* and gk275304rf, respectively, both enhance the CAN positioning defect of mig-2(gm38gf). I) The ral-1(gk628801rf) allele that abrogates RAL-1 signaling also enhances the CAN positioning defect of mig-2(gm38gf). Throughout, each data point is shown. The median is shown by a line, the box indicates 25% and 75% confidence intervals, and bars indicate outlying data. **** represents P<0.0001, *** P<0.001, ** P<0.01, and * P<0.05.

Figure 2:. The role of RalGEF>Ral signaling is not specific to sensitizing genetic background.

A) mig-2(gm103gf) conferred stronger migration defects than did mig-2(gm38gf). B) Deletion allele rgl-1(tm2255) failed to enhance the defect of gm103gf animals. C, E) Moderate and strong alleles of vab-8, gm84 and e1017, respectively confer CAN migration defects. D, F) The rgl-1 deletion allele ok1921 enhanced the moderate gm84 but not the strong e1017 alleles of vab-8, respectively. The median is shown by a line, the box indicates 25% and 75% confidence intervals, and bars indicate outlying data. **** represents P<0.0001 and *** P<0.001.

Figure 3:. RAL-1/Ral is expressed in the CANs.

Confocal micrographs. A) CAN labeled in green by otIs33[P_kal-1::gfp]. B) red-tagged RAL-1, ral-1(re218[mKate2::2xHA::RAL-1]). C) Merged images of ral-1(re218[mKate2::RAL-1]); otIs33[Pkal-1::gfp]. Arrows indicates the CAN. Scale bars = 10 μm.

Figure 4:. RGL-1/RalGEF>RAL-1/Ral signaling functions cell autonomously to regulate can positioning.

A, B, C) Photomicrographs of CAN-specific expression of rescuing mKate2::RAL-1 in an animal of genotype reSi8[P_{ceh-23_L}::mK2::ral-1]; ral-1(gk628801rf); otIs33[P_kal-1::gfp] using DIC, green and red channels, respectively. D,E,F) Photomicrographs of CAN-specific expression of rescuing mKate2::RGL-1 in an animal of genotype reSi14[P_{ceh-23_L}::mK2::rgl-1]; rgl-1(gk275304rf); otIs33[Pkal-1::gfp] using DIC, green and red channels, respectively. G) CAN-specific expression of mK2::RAL-1 partially rescues the enhanced CAN migration phenotype of gk628801; gm38gf. H, I) CAN-specific expression of mK2::RGL-1 partially rescues the enhanced CAN migration phenotype of gk275304; gm38gf. * represents P<0.05.

Figure 5:. Deletion of RGL-1/RalGEF enhanced defects in general nervous system function and development.

A) Measurement of radial locomotion (see Methods) shows that genetic interactions controlling locomotion reflect those observed for CAN positioning in Figure 1. mig-2(gm103gf) is more severely defective in locomotion than is mig-2(gm38gf), and that rgl-1(tm2255) enhances gm38 but not gm103. N=30 for each strain. B) A marker of DD and VD axons, juIs76[P_unc-25::GFP], revealed that dorsoventral axon guidance was defective in mig-2(gm38gf) animals and was enhanced by rgl-1(tm2255). C, D, E) Epifluorescence photomicrographs of circumferential axon migration in wild-type, gm38 and tm2255 gm38 animals, respectively. Scale bar = 10 μm. **** represents P<0.0001.

Figure 6:. RAL-1/Ral functions as a permissive cue while LET-60/Ras functions as an instructive cue in CAN migration.

A,B) Constitutively active ral-1(re160gf) and wild-type ral-1(re218), both tagged with the same fluorescent protein and epitope, failed to enhance the CAN positioning defect conferred by mig-2(gm38gf). C) let-60(n1046gf), which causes constitutive activation, enhances the CAN positioning defect of mig-2(gm38gf). D) Deletion of rap-1, rap-1(tm861), enhanced the positioning defect of gm38gf. E) ral-1(gk628801rf) and rap-1(tm861) together did not enhance the positioning defect of CANs compared to their individual effects. F) rap-1(re180gf) failed to enhance the CAN positioning defect. The median is shown by a line, the box indicates 25% and 75% confidence intervals, and bars indicate outlying data. **** represents P<0.0001, *** P<0.001, ** P<0.01, and * P<0.05. H) A model for the function of the parallel RAP-1/Rap1>RGL-1/RalGEF>RAL-1/Ral and LET-60/Ras signals controlling migration of the CANs, as well as other parallel signals.

Figure 7:. The RLBP-1/RalBP1 canonical effector of Ral functions in the opposite role as RAL-1 in positioning of CANs.

A) Reduction of function of putative Ral effector sec-5 function did not alter CAN positioning in the gm38gf background. Non-Green homozygous sec-5(pk2357rf) animals from heterozygous mothers in which the sec-5 mutation was balanced by GFP-labelled chromosomal inversion, mIn1. B) Deletion of putative Ral effector Exo84, exoc-8(ok2523), did not alter CAN positioning in the gm38gf background. C) Deletion of putative Ral effector RalBP1 by rlbp-1(tm3665) unexpectedly suppressed the migration defect of gm38gf. The median is shown by a line, the box indicates 25% and 75% confidence intervals, and bars indicate outlying data. **** represents P<0.0001, *** P<0.001, ** P<0.01, and * P<0.05. D) A model for the function of parallel Rap1^RAP-1>RalGEF^RGL-1>Ral^RAL-1 and Ras^LET-60 signals controlling migration of the CANs, as well as other parallel signals.