RESEARCH ARTICLE 4433
Development 138, 4433-4442 (2011) doi:10.1242/dev.068841 © 2011. Published by The Company of Biologists Ltd UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans Adam D. Norris and Erik A. Lundquist*
SUMMARY The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-of- function unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.
KEY WORDS: Filopodia, Growth cone, Lamellipodia, Protrusion, UNC-6/netrin
INTRODUCTION to an attractive response by the growth cone. In neurons expressing Developing axons are led by growth cones, dynamic actin-based both UNC-5 and DCC, netrin binding leads primarily to UNC-5 structures that sense and respond to extracellular cues, driving the alone or UNC-5 and DCC signaling, both of which mediate a forward motion of the axon (Mortimer et al., 2008; Tessier-Lavigne repulsive response (Hong et al., 1999; MacNeil et al., 2009). and Goodman, 1996). Growth cones consist of lammelipodial Whereas the role of UNC-6/netrin and its receptors have been protrusions of a branched actin meshwork and filopodial studied extensively in axon pathfinding endpoint analyses and in protrusions of bundled actin filaments, which are both involved in vitro growth cone experiments, little is known about how they the proper growth of an axon to its target destination (Gallo and affect the growth cone in vivo during growth cone outgrowth. Letourneau, 2004; Pak et al., 2008; Zhou and Cohan, 2004). Previous studies have shown that UNC-6/netrin can affect the The laminin-like UNC-6/netrin guidance cue and its receptors polarity of growth cone protrusions in C. elegans (Adler et al., UNC-40/DCC (deleted in colorectal carcinoma) and UNC-5 have 2006), and the morphology of the growth cone in neurons in vitro been widely implicated in axon pathfinding in mouse, Drosophila, (Shekarabi and Kennedy, 2002; Shekarabi et al., 2005). This led us C. elegans and other organisms (Chan et al., 1996; Hong et al., to speculate that UNC-6/netrin and its receptors may play an 1999; Leonardo et al., 1997; Montell, 1999; Pan et al., 2010; important role in modulating both the extent and the polarity of Shekarabi and Kennedy, 2002; Moore et al., 2007). In C. elegans, protrusion in C. elegans growth cones. UNC-6 is secreted by ventral cells and is necessary for guidance of We endeavored to understand how UNC-6/netrin and its circumferential neurons (Ishii et al., 1992). The receptor UNC-40 receptors UNC-40/DCC and UNC-5 affect axon pathfinding, is expressed in growth cones and mediates an attractive response growth cone morphology and dynamics in circumferential to UNC-6, driving the ventral guidance of circumferential axons, neurons in C. elegans, using methods we have previously while the receptor UNC-5 is expressed in dorsally guided growth developed that allow in vivo analysis of C. elegans growth cones and mediates a repulsive response to UNC-6, driving the cones. We show here that UNC-6/netrin and its receptors UNC- dorsal guidance of circumferential axons (Hedgecock et al., 1990; 40/DCC and UNC-5 are involved in both polarity of growth Hong et al., 1999). cone protrusiveness as well as in modulating the extent of Vertebrate DCC and UNC-5 both respond to netrin by the protrusion. Indeed, these molecules affected filopodial dynamics formation of receptor dimers. In neurons expressing only DCC, independently of their polarity of protrusion, suggesting that netrin binding causes the formation of an DCC homodimer, leading polarity of protrusion and extent of protrusion might be governed at least in part by separate mechanisms and that growth cone guidance in vivo involves a combination of the two. Our results also suggest that UNC-5, UNC-6 and UNC-40 regulate a balance Programs in Genetics and Molecular, Cellular, and Developmental Biology, Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, of protrusion within a growth cone, with UNC-6 driving Lawrence, KS 66045, USA. protrusion via UNC-40 and inhibiting protrusion via UNC-5. This balance might establish a dorsal-ventral gradient of *Author for correspondence ([email protected]) protrusion across the growth cone, which in part might underlie
Accepted 1 August 2011 growth cone polarity and directed outgrowth. DEVELOPMENT 4434 RESEARCH ARTICLE Development 138 (20)
MATERIALS AND METHODS Growth cone time-lapse imaging Genetic methods VD growth cones were imaged as previously described (Norris et al., 2009). Experiments performed at 20°C using standard C. elegans techniques Briefly, animals harboring the juIs76 [unc-25::gfp] transgene were selected (Brenner, 1974). These mutations and transgenics were used: X, unc- 16 hours post-hatching at 20°C and placed on a 2% agarose pad with a drop 6(ev400 and e78), lqIs170 [rgef-1::vab-10ABD::gfp]; I, unc-40(n324 and of 10 mM muscimol (Sigma-Aldrich, St Louis, MO, USA) in M9 (Weinkove e1430), unc-73(rh40); II, juIs76 [unc-25::gfp]; IV, kyIs179 [unc-86::gfp], et al., 2008), which was allowed to evaporate for 3-4 minutes before placing unc-5(e53 and e152); ?, lqIs128 [unc-25::myr::unc-40], lqIs129 [unc- coverslip on top of the animals on the pad. Growth cones were imaged with 25::myr::unc-40], lqIs164 [myo-3::cfp], lqIs182 [unc-86::myr::gfp] and a Qimaging Rolera mGi EMCCD camera using a very short exposure time lhIs6 [unc-25::mCherry]. Extrachromosomal arrays were attained by (~30 mseconds) on a Leica DMR microscope. Images were acquired at injection into the germline, and then integrated into the genome via intervals of 120 seconds, with total duration of time lapse ranging from 20 standard techniques (Mello and Fire, 1995). Double mutants with unc-6, to 60 minutes. HSN growth cone time-lapse imaging was performed unc-5 and unc-40 were confirmed by the uncoordinated movement identically, except that animals were selected at 19 hours post hatching. phenotype, axon pathfinding phenotype and PCR genotyping. Growth cone protrusions were scored as previously described (Norris et al., 2009). Dynamic projections less than 0.5 m in width emanating from Imaging of axon guidance defects the growth cone were scored as filopodia; protrusions greater than 0.5 m VD neurons were visualized with an unc-25::gfp (Jin et al., 1999) or unc- were considered lamellipodial extensions; and protrusions that were static 25::mCherry transgene, expressed in all GABAergic neurons, including the were not considered filopodia. Filopodia were considered to have 16 VDs. VD axons were considered defective if the axon failed to reach disappeared at the point when there was no longer a visible a protrusion of the dorsal nerve cord or if it branched or turned at an angle greater than 45° less than 0.5 m protruding from the membrane at the location where the before reaching the dorsal nerve cord. To determine severity of defects, a filopodium had once been. Filopodia length and growth cone area were myo-3::cfp transgene was introduced to mark the ventral and dorsal muscle measured using ImageJ software. Filopodia length was determined by quadrants. Using an unc-25::mCherry construct we could then determine drawing a straight line from the base where the filopodium originates on whether an the trajectory of an axon went awry before or after reaching the the edge of the peripheral membrane to the tip of the filopodium. Polarity dorsal edge of the ventral muscle quadrant. of filopodia protrusion was determined using ImageJ and dividing the HSNs were visualized with an unc-86::gfp transgene (Shen and growth cone into roughly four equally sized quadrants (dorsal-anterior, Bargmann, 2003), which is expressed in numerous neurons, including the dorsal-posterior, ventral-anterior and ventral-posterior) and determining two HSNs. HSN axons were considered defective if the axon failed to from which quadrant each filopodium emanated (see Fig. 7B for example). reach the ventral nerve cord, or if it branched or turned at an angle greater Significance of difference was determined in all cases by a two-sided t-test than 45° before reaching the ventral nerve cord. with unequal variance or a Fisher’s exact test.
Fig. 1. Netrin signaling mutants cause axon pathfinding defects. (A)Percentage of VD/DD axons with pathfinding defects. (B-D)Fluorescent micrograph of L4 VD/DD axons; (B) normal pathfinding in wild-type axons (arrows) that reach the dorsal nerve cord (arrowheads). (C)Misguided and prematurely stopped axons (arrow) that do not reach the DNC (arrowheads) in unc-6(ev400). (D)An ectopic axon branch in unc-5(e53) (arrow). Arrowheads indicate axons. (E)Percentage of VD/DD axons with obvious ectopic axon branches. (F)Percentage of HSN axons with pathfinding defects. (G,H)Fluorescent micrographs of wild-type and unc-6(ev400) HSN axons; (G) normal pathfinding in wild type; and (H) failure to migrate ventrally in unc-6(ev400). Scale bar: 5m. Asterisks indicate significant difference between genotypes (P<0.01) determined by Fisher’s exact test, and all genotypes were significantly different from wild type. Error bars represent 2ϫ standard error of the proportion. DEVELOPMENT Netrin and the growth cone RESEARCH ARTICLE 4435
VAB-10ABD::GFP for F-actin visualization 1A,F). These results are consistent with previous findings that F-actin was visualized by imaging growth cones expressing the F-actin UNC-6/netrin and UNC-40/DCC, but not UNC-5, are required for binding domain of spectraplakin VAB-10 fused to GFP, which has previously ventral migration (Hedgecock et al., 1990). been used to monitor F-actin in other C. elegans cells (Bosher et al., 2003; Patel et al., 2008). We compared localization of this construct to the UNC-5 and UNC-6 inhibit growth cone protrusion localization of a cytoplasmic mCherry to control for nonspecific factors such Although it is established that UNC-6/netrin signaling affects as variable growth cone size and shape. Asymmetric accumulation was quantified with line scans through the growth cone on the dorsal-ventral axis circumferential axon guidance, the effects of these molecules on using NIH Image (see Fig. 8). Five line scans were made through each the growth cone during outgrowth in vivo are less well understood. growth cone, and the average pixel intensity of the green channel was We imaged the growth cones of the VD motoneurons that are divided by the average intensity of the red channel to yield a relative intensity repelled from UNC-6/netrin over time as they extended. VD ratio indicating the level of enrichment of the VAB-10ABD::GFP construct. growth cones were imaged in animals at 16 hours post-hatching at a time when the VD growth cones have begun their commissural RESULTS migrations (see Materials and methods for imaging methods). We UNC-5, UNC-6 and UNC-40 control dorsal-ventral measured growth cone area as well as the dynamic parameters of axon pathfinding filopodia initiation rate, filopodia duration and maximal filopodial In C. elegans, unc-6 and unc-40 mutants display guidance defects length (see Materials and methods for parameter measurement). in both ventrally directed and dorsally directed circumferential Wild-type VD growth cones were dynamic (see Movie 1 in the axons, and unc-5 mutants display defective pathfinding in dorsally supplementary material) and had an average area of 6.7 m2 (Fig. directed circumferential axons (Hedgecock et al., 1990). We 2A). Wild-type growth cones also displayed multiple dynamic confirmed and quantified defects in unc-5, unc-6 and unc-40 filopodia that formed at an average rate of 0.26 per minute (one mutants in the dorsally directed VD motoneurons that are repelled initiation event every 3.8 minutes) (Fig. 2B,F). The average wild- from UNC-6, and in the ventrally directed HSN axons that are type filopodium had a duration of 4.9 minutes (between the time it attracted to UNC-6 (Fig. 1). Consistent with previous results, unc- was first noticeable to the time it was no longer apparent) (Fig. 2C) 5 and unc-6 mutants displayed a nearly complete failure of VD and had an average maximal length of 0.96 m (Fig. 2D). These axons to reach the dorsal nerve cord, whereas unc-40 mutants had parameters are similar to those previously observed for wild-type a weaker effect on VD axon pathfinding (Fig. 1A-D). By contrast, VD growth cones (Norris et al., 2009). unc-6 and unc-40 mutants had strong HSN axon guidance defects, To understand how axonal repulsion relates to growth cone whereas unc-5 mutants did not (Fig. 1F-H). The unc-6(e78) allele, morphology during outgrowth, we analyzed VD growth cones in which results in a cysteine to tyrosine substitution in domain V-3 unc-5 mutants. unc-5 caused an increase in growth cone that disrupts interaction with UNC-5 but not UNC-40/DCC (Lim protrusiveness in VD growth cones (see Movie 2 in the and Wadsworth, 2002), more strongly affected dorsal repulsive supplementary material). Growth cone area was significantly growth than ventral attractive growth, as previously reported (Fig. increased (Fig. 2A,G), as was filopodia duration (Fig. 2C). Average
Fig. 2. Netrin signaling mutants affect VD growth cone morphology and filopodial dynamics. (A-D)Quantification of filopodia dynamics in VD growth cones. (A)Growth cone area inm2. (B)Filopodia formation rate, in initiation events per minute. (C)Duration of filopodia once formed, in minutes. (D)Maximum filopodia length, inm. (E)Percentage of filopodia greater than the wild-type average + 2 standard deviations in length. Error bars represent 2ϫ standard error of the mean; asterisks indicate the significant difference between wild-type and the mutant genotype (P<0.01) determined by two-sided t-test with unequal variance. (F,G)Timelapse series of live growth cones, taken at 2 minutes per frame. (F)A wild-type VD growth cone. (G)An unc-5(e53) growth cone. Arrowheads indicate representative filopodia. Scale bar: 5m. DEVELOPMENT 4436 RESEARCH ARTICLE Development 138 (20) maximal filopodial length was also increased significantly (Fig. maximal filopodial length compared with wild type (Fig. 2D). This 2D). However, filopodial length appeared biphasic in unc-5 is surprising given that UNC-40 is thought to cooperate with UNC- mutants, with many filopodia that were comparable with wild-type 5 in repulsive axon guidance. length and some that grew very long (greater than two standard To probe the role of UNC-40 in VD growth cone dynamics in deviations from average wild-type length) (Fig. 2E,G; see Movie 1 more detail, we constructed a constitutively active version of UNC- in the supplementary material). Some of these long filopodia were 40, based upon Gitai et al. (Gitai et al., 2003); by adding an N- observed to endure throughout the growth cone imaging period, terminal myristoylation signal to the cytoplasmic domain of UNC- suggesting that they are very stable growth cone protrusions not 40. MYR::UNC-40 lacks the transmembrane domain and the observed in wild type. These data indicate that UNC-5 is normally extracellular domains, which function to inhibit dimerization in the required to limit the extent of protrusion of growth cones that are absence of UNC-6/netrin. MYR::UNC-40 is targeted to membranes repelled from UNC-6. unc-5 mutants did not affect the rate of by the myristoylation sequence and is thought to be constitutively filopodia formation (Fig. 2B), suggesting that UNC-5 might affect active even in the absence of UNC-6/netrin. filopodial dynamics after a filopodium has been formed. Expression of myr::unc-40 was driven in the VD neurons using Surprisingly, the unc-6(ev400) null mutation had no significant the unc-25 promoter. In a wild-type background, MYR::UNC-40 effect on any of the VD growth cone parameters we measured (Fig. caused a marked decrease in VD growth cone protrusiveness (see 2A-E). However, the unc-6(e78) mutation that specifically disrupts Movie 3 in the supplementary material). MYR::UNC-40 growth the repulsive, UNC-5-dependent role of UNC-6 (Lim and cones were small [6.8 m2 in wild type compared with 4.6 m2 in Wadsworth, 2002), resembled unc-5 mutants in all parameters lqIs128[myr::unc-40]; P<0.01 (Fig. 2A)] with few filopodial measured (increased growth cone area, increased filopodial protrusions, and exhibited a ‘treadmilling’ behavior in which there duration and unusually long filopodia with no effect on filopodia was some movement around the edges of the growth cone but very initiation rate) (Fig. 2A-E). That these effects are reduced in the little growth cone advance (see Movie 3 in the supplementary unc-6 null suggests that in unc-6(e78), increased growth cone material). MYR::UNC-40 growth cones exhibited a decrease in protrusiveness was due to a non-UNC-5 mediated role of UNC-6, filopodial formation rate (0.26 per minute in wild-type compared possibly interaction with UNC-40. Indeed, as we show below, loss with 0.16 per minute in lqIs128[myr::unc-40]; P<0.01) (Fig. 2B). of UNC-40 suppressed the increased filopodial dynamics in unc-5 When filopodia did form, they had reduced duration (Fig. 2C) and mutants and in unc-6(e78), indicating that UNC-40 is required for reduced maximal length (Fig. 2D). These data suggest that UNC-40 increased growth cone protrusion in these mutants. normally has a role in inhibition of growth cone protrusion. In contrast to loss of function of unc-5 and unc-6(e78), myr::unc-40 Constitutively-active MYR::UNC-40 inhibits VD also affected filopodia initiation rate. Thus, UNC-40 might also be growth cone protrusion involved in inhibition of filopodial initiation. Alternatively, filopodia Two putative null mutations in unc-40, e1430 and n324, had very might form at a normal rate in myr::unc-40 but are inhibited before little effect on VD growth cone dynamics and morphology (Fig. they are visible in our imaging conditions, which might not be able 2A-E), with the only significant effect being a reduction in to distinguish short or transient filopodia. Consistent with these
Fig. 3. MYR::UNC-40-mediated inhibition of VD growth cone protrusion requires UNC-5. (A-D)Quantification of filopodia dynamics in VD growth cones as described in Fig. 2. Error bars represent 2ϫ standard error of the mean; asterisks indicate significant difference between lqIs128[myr::unc-40] and the double mutant (P<0.01) determined by two-sided t-test with unequal variance. (E,F)Fluorescent micrographs of typical mutant VD growth cones, with a line tracing of the growth cone perimeter and filopodial protrusions shown on the right. Scale bar: 5m. DEVELOPMENT Netrin and the growth cone RESEARCH ARTICLE 4437 defects in growth cone morphology, myr::unc-40 caused defects in excess protrusion in unc-5 mutants was due in part to slow VD and DD dorsal axon pathfinding, as determined by end-point advance. We think this is unlikely because MYR::UNC-40 growth analysis (55% defective axons in lqIs128[myr::unc-40]) (Fig. 1A). cones, which also displayed slow advance, had reduced protrusion; That loss of unc-5 and activation of myr::unc-40 had opposite effects and because unc-5 loss of function suppressed the inhibition of on growth cone protrusiveness indicate that these are bona fide protrusion of MYR::UNC-40 and restored protrusion to unc-5-like effects of the molecules on the growth cone and not due to a general levels, indicating that UNC-5 was directly required for inhibiting perturbation of growth cone function. protrusion.
Constitutively active MYR::UNC-40 requires UNC-5 VD growth cone over-protrusive phenotypes of to inhibit VD protrusion unc-5 and unc-6(e78) are suppressed by unc-40 We next tested whether the MYR::UNC-40-mediated reduction in mutation growth cone protrusion was dependent on the presence of other unc-5 null and unc-6(e78) mutants but not unc-6(ev400) null netrin signaling components. We found that the MYR::UNC-40 mutants displayed increased growth cone protrusion, suggesting a phenotype was not dependent on UNC-6, as unc-6(ev400); myr::unc- role of UNC-6 in stimulating VD growth cone protrusion in the 40 double mutant VD growth cones resembled myr::unc-40 alone absence of UNC-5. We speculated that this might involve UNC-40. (Fig. 3A-D). This result is consistent with the idea that MYR::UNC- We constructed unc-5; unc-40 double mutants and found that they 40 is an UNC-6-independent, constitutively active molecule. did not exhibit the over-protrusive phenotypes seen in the unc-5 MYR::UNC-40 was also not dependent on endogenous UNC-40 single mutants (Fig. 4): growth cone size, filopodial duration and (Fig. 3A-D). However, MYR::UNC-40 inhibition of protrusion was filopodia length (Fig. 4A-C) all returned to near-wild-type levels dependent upon UNC-5, as unc-5(e53) suppressed the effects of in the double mutants using two distinct null alleles of unc-40. myr::unc-40 (Fig. 3A-F, see Movie 4 in the supplementary material). Similarly, unc-40(n324) was required for the over-protrusive unc-5(e53) restored growth cone area, filopodia initiation rate, growth cones of unc-6(e78) mutants, as unc-6(e78); unc-40(n324) duration and maximal length (Fig. 3A-D). unc-5(e53); myr::unc-40 double mutants had near wild-type levels of protrusion (Fig. 4A- had a protrusion comparable with or greater than that of wild type, C). These results suggest that UNC-40 and UNC-6 have roles in and in fact resembled the excessive protrusion seen in unc-5 mutants stimulating VD growth cone protrusion in addition to roles in alone (e.g. filopodial duration was increased in unc-5(e53); inhibition of protrusion along with UNC-5, as demonstrated by the lqIs128[myr::unc-40] compared with wild type; Fig. 3C). MYR::UNC-40 results above. The above findings might also These results suggest that MYR::UNC-40 requires UNC-5 to explain why unc-6-null mutants and unc-40 mutants display less inhibit growth cone protrusion, consistent with previous results severe growth cone protrusion defects than unc-5 mutants (i.e. suggesting that an UNC-40/UNC-5 heterodimer mediates growth UNC-6 and UNC-40 are required both to stimulate and to inhibit away from UNC-6/netrin. These results also suggest that formation protrusion, and when they are gone an equilibrium between the two of a putative MYR::UNC-40/UNC-5 heterodimer is independent is maintained). of UNC-6/netrin, as would be expected of a ligand-independent This balance of protrusion in the growth cone leads to the idea constitutively active molecule. that protrusion might be asymetrically affected across the growth Previous studies indicated that stationary or slow-moving growth cone, which might explain in part growth cone polarity and cones often excess protrusion (Knobel et al., 1999), and we found directed outgrowth. Indeed, in wild-type VD growth cones, that unc-5 mutant growth cones exhibited slow advance. Possibly, dorsally directed filopodia had longer duration and length than
Fig. 4. unc-5 mutant excessive growth cone protrusion requires UNC-40/DCC. (A-C)Quantification of filopodia dynamics in VD growth cones as described in Fig. 2. Error bars represent 2ϫ standard error of the mean; asterisks indicate significant difference between wild type and the mutant (P<0.01); + indicates significant difference between double mutant and its respective protrusion-stimulating mutant (P<0.01) determined by two-sided t-test with unequal variance. (D,E)Fluorescent micrographs of typical mutant VD growth cones, with a line tracing of the growth cone perimeter and filopodial protrusions shown on the right. Scale bar: 5m. DEVELOPMENT 4438 RESEARCH ARTICLE Development 138 (20) ventrally directed filopodia, indicating that protrusion is greater in the direction furthest from the source of UNC-6/netrin (6.6 minutes dorsal versus 4.8 minutes ventral; P<0.02; n>50 filopodia; and 1.0 m dorsal versus 0.8 m ventral; P<0.02; n>50 filopodia).
Axon pathfinding defects of unc-5 mutants are reduced by unc-40 mutation Previous studies indicated that UNC-5 alone signaling mediates repulsion while close to the UNC-6/netrin source, whereas UNC- 5 and UNC-40 signaling mediates repulsion at locations more distant from the UNC-6/netrin source (MacNeil et al., 2009). We labeled the ventral and dorsal muscle quadrants with CFP and the VD axons with mCherry, allowing us to quantify the number of axon trajectories that go awry before passing the ventral muscle quadrant, close to the normal source of UNC-6/netrin (Fig. 5) (Ishii et al., 1992). Indeed, most VD axon pathfinding defects in unc-40 mutants occurred after the axon passed the ventral muscle quadrant [>90% in unc-40(n324)] (Fig. 5A), whereas most unc-5(e53) null and unc-6(e78) defects occurred in or before the ventral muscle quadrant, with some axons apparently not emerging from the ventral nerve cord (Fig. 5A,B). unc-5(e53) and unc-6(e78) mutants also had significantly more severe defects than the unc-6(ev400) null, similar to the effects on growth cone protrusion. unc- 40(n324); unc-5(e53) double mutants were less severe than unc- 5(e53) alone, and had guidance defects of comparable severity to unc-6(ev400) alone. These data indicate that in unc-5 mutant VD Fig. 5. Severity of unc-5(e53) axon pathfinding defects is growth cones, UNC-40/DCC may be mediating an attractive unc-40. response to UNC-6/netrin, causing these axons to become attracted mitigated by loss of (A)Percentage of VD axons that have pathfinding errors by the time they reach the dorsal edge of the ventral to, rather than repelled from, UNC-6/netrin, and thus misguided muscle quadrant. Error bars represent 2ϫ standard error of the mean; sooner than in the complete absence of UNC-6/netrin. asterisks indicate a significant difference between wild type and the mutant (P<0.01); + indicates a significant difference between unc- Netrin signaling component mutants modulate 5(e53) alone and the unc-5(e53); unc-40(n324) double mutant (P<0.01) extent of protrusion of HSN growth cones determined by two-sided t-test with unequal variance. To study the roles of UNC-6 and UNC-40 in growth cones that are (B,C)Representative images showing VD axons (arrows) with mCherry attracted to UNC-6/netrin, we turned to the HSN neurons. The expression (red) after their complete outgrowth in L4 animals. Ventral bilateral HSN cell bodies reside laterally just behind the and dorsal muscle quadrants with CFP expression are blue. In unc- hermaphrodite vulva in the midsection of the animal. The HSN 5(e53) most axons do not advance beyond the ventral muscle axons extend ventrally to the ventral nerve cord and then anteriorly quadrants, whereas in unc-5(e53); unc-40(n324), many axons extend past the ventral quadrants. Scale bar: 5m. to innervate the vulva. As previously reported, the axons of the HSN neurons require UNC-6 and UNC-40 for their ventralward migration (Fig. 1F-H) (Adler et al., 2006). In unc-6 and unc-40 mutants, the required for robust HSN growth cone protrusion. As expected, the HSN axons often extend laterally instead of ventrally. The HSN unc-6(e78) mutation, which specifically attenuates UNC-5-mediated displays a prominent growth cone at 18-19 hours post-hatching in effects of UNC-6, had no effect on HSN growth cones (Fig. 6). the late L1 stage (see Movie 5 in the supplementary material). At this We drove the expression of MYR::UNC-40 in the HSNs using time, the growth cone resembles a thickened protrusion from the the unc-86 promoter. In contrast to inhibiting protrusion in the ventral region of the cell body with multiple filopodial protrusions, VDs, MYR::UNC-40 caused an increase in protrusiveness in the as previously reported (Adler et al., 2006). Over the next 12 hours HSNs (see Movie 7 in the supplementary material), with an into the mid L4 larval stage, the HSN growth cone remains dynamic increase in growth cone size, filopodia duration and length (Fig. 6). and migrates ventrally to the ventral nerve cord. We analyzed HSN This is the opposite effect seen with MYR::UNC-40 in the VDs, growth cones in time lapse at 19 hours post-hatching. At this time and suggests that in axons that do not express UNC-5, the the wild-type HSN growth cone is on average 11.2 m2 in size, with MYR::UNC-40 molecules form an attractive complex that filopodia that form at an average rate of one every 4.4 minutes and stimulates growth cone protrusion. endure for 5.2 minutes, with a maximal filopodial length of 0.9 m Considered with the results in the VD growth cones, these data (Fig. 6 and see Movie 5 in the supplementary material). The HSN suggest that UNC-6/netrin and it receptors UNC-40/DCC and UNC- growth cone is larger, but all dynamic filopodial characteristics are 5 control the extent of growth cone protrusion, including growth cone similar to those of the VD growth cones. size and the maximal length and duration of growth cone filopodia. unc-40- and unc-6-null mutant HSN growth cones exhibited a Inhibition versus stimulation of protrusion were correlated with decrease in protrusiveness (see Movie 6 in the supplementary repulsion versus attraction in response to UNC-6/netrin. However, in material), with a decrease in growth cone, filopodial duration and the VD growth cones, unc-6 mutants had only weak effects on growth filopodia length (Fig. 6A-D). unc-6(ev400)-null mutants but not unc- cone protrusiveness but very strong VD axon guidance defects, 40 mutants also exhibited a decrease in filopodia formation rate (Fig. indicating that regulation of growth cone protrusion is not the only
6B). These results indicate that UNC-6/netrin and UNC-40/DCC are role of UNC-6/netrin in growth cones during axon pathfinding. DEVELOPMENT Netrin and the growth cone RESEARCH ARTICLE 4439
Fig. 6. Netrin signaling mutants cause HSN growth cone morphology and dynamic defects. (A-E)Quantification of filopodia dynamics in HSN growth cones as described in Fig. 2. Error bars represent 2ϫ standard error of the mean and asterisks indicate significant difference between wild type and the mutant (P<0.01) determined by two-sided t-test with unequal variance. (F)A typical wild-type HSN growth cone that protrudes ventrally from the cell body. (G)A typical unc- 40(n324) HSN growth cone, which often protrudes anteriorly and laterally rather than ventrally from the cell body. Arrowheads indicate growth cones. Scale bar: 5m.
UNC-5, UNC-6 and UNC-40 affect polarity of from the anterior of the cell body. Loss of UNC-5 did not affect growth cone filopodia protrusion dorsal-ventral polarity, which is consistent with the observation Wild-type VD and HSN growth cones display highly polarized that UNC-5 is not expressed in the HSN (Fig. 7E,H). However, filopodial protrusion: in VD growth cones, most filopodia loss of UNC-40/DCC or UNC-6/netrin abolished the ventral protrude dorsally away from UNC-6 (Fig. 7A,B,D); and in HSN polarity of filopodia extension (Fig. 7E,G,H), and the growth growth cones, most filopodia protrude ventrally toward UNC-6 cone often extended anterolaterally instead of ventrally (Fig. (Fig. 7E,F,H). We next determined whether orientation of 7G). These results are consistent with previous results showing filopodial protrusion was altered in unc-5, unc-6 and unc-40 that UNC-6 localizes UNC-40 to the ventral surface of the HSN mutants. Previous studies indicated that UNC-6 was required for and, in an unc-6 mutant, UNC-40 localizes uniformly around the polarized distribution of UNC-40 and MIG-10/lamellipodin on cell body and protrusions extend from around the cell body (Xu the ventral region of the HSN neuron and that unc-6 and unc-40 et al., 2009). These results indicate that UNC-5, UNC-6 and mutant HSN neurons displayed a loss of polarized ventral UNC-40 control the polarity of filopodial protrusion from protrusion (Adler et al., 2006; Quinn et al., 2006; Quinn et al., growth cones that are repelled (VDs) or attracted (HSNs) to 2008; Xu et al., 2009). To quantify polarity of filopodial UNC-6/netrin. protrusion, we divided the growth cones into four quadrants along the AP and DV axes, based upon the orientation of the UNC-5 and UNC-6 control polarity of growth cone animal, regardless of the direction that the growth cone was filamentous actin navigating (some VD growth cones in mutants were misguided Growth cone protrusion, including the lamellipodial growth cone and were migrating laterally or even ventrally; and many HSN body and filopodial extensions, involves dynamic regulation of the growth cones protruded laterally rather than ventrally). In wild actin cytoskeleton (Quinn and Wadsworth, 2008). In the VDs and type, VD neurons growing along naked epidermis, the majority the PQR dendrite growth cones, the actin regulators Arp2/3, UNC- of filopodia (70%) extended from the dorsal half of the growth 115/abLIM and UNC-34/enabled are required for robust filopodial cone (Fig. 7A,B,D). unc-5- and unc-6-null mutations abolished extensions (Norris et al., 2009). We assayed filamentous actin (F- this polarity such that filopodia protruded nearly equally in the actin) in the VD growth cones, using a construct in which the F- dorsal and ventral directions (Fig. 7A,C,D). During the stage at actin binding domain of the spectraplakin VAB-10 is fused to GFP which we imaged VD growth cones, unc-40-null mutations had that has been used to monitor F-actin in other cells in living C. no significant effect on dorsal ventral polarity of VD growth elegans (Bosher et al., 2003; Patel et al., 2008). In wild-type VD cone filopodial protrusion, nor did MYR::UNC-40. Anterior- growth cones, this VAB-10ABD::GFP construct localized posterior VD filopodial protrusion was not significantly different preferentially to the dorsal leading edge of the lamellipodia and in in wild-type or any mutant (Fig. 7D). filopodial protrusions (Fig. 8A,B,E). On average, VAB- In wild-type HSN growth cones, the majority of filopodia 10ABD::GFP showed 1.22-fold more accumulation in the dorsal extended from the ventral half of the growth cone, towards their half of growth cones compared with the ventral halves (Fig. 8G). target in the VNC (Fig. 7E,F,H). In HSN growth cones of all In unc-6(ev400)-null mutants and unc-5(e53)-null mutants, VAB- genotypes, there was a bias toward anterior versus posterior 10ABD::GFP no longer showed a preferential dorsal leading edge
protrusion, as the HSN growth cone generally extends ventrally accumulation (Fig. 8C,D,G). Rather, VAB-10ABD::GFP DEVELOPMENT 4440 RESEARCH ARTICLE Development 138 (20)
Fig. 7. Netrin signaling mutants cause growth cone polarity defects. (A)Percentage of filopodia protruding dorsally from VD growth cones. The x-axis is set at 50%, such that bars extending above the x-axis represent percentages above 50%, and bars that extend below the axis represent percentages below 50%. Asterisks indicate a significant difference between wild type and the mutant (P<0.01) determined by two-sided t-test with unequal variance. (B,C)Representative images of wild-type and unc- 5(e53) growth cones, with a line drawing of the growth cone and filopodial perimeter on the right. Scale bar: 5m for B,C,F,G. Dashed gray lines in B delineate dorsal and ventral boundaries of the growth cone; solid lines indicate the dorsal-ventral and anterior-posterior boundaries used for scoring filopodia polarity. (D)Graphical representations of relative filopodial protrusion from each quadrant of the VD growth cones (dorsal left, dorsal right, ventral right, ventral left), generated by the Spoke12 program (M. Tourtellot, University of Kansas). Dorsal is upwards and anterior is towards the left. (E)Percentage of filopodia protruding dorsally from HSN growth cones, as described in A. (F,G)Representative images of wild-type and unc- 40(n324) growth cones, with arrows marking growth cone protrusions. (H)Relative filopodial protrusion from each quadrant of HSN growth cones as described in D.
distribution was randomized across the growth cone (Fig. 8E) and guidance in unc-6 mutants suggests that other guidance signals, in some growth cones showed a ventral bias (Fig. 8C). In unc- such as UNC-129 or SLT-1 may still be affecting the migration of 6(ev400), F-actin was often seen to accumulate at both the dorsal VD and HSN growth cones (e.g. SLT-1 preventing HSN axons and ventral margins of the growth cone (Fig. 8F). F-actin from migrating dorsally). accumulated in filopodial protrusions at the margin, so this might represent randomized filopodial protrusion, as seen in the growth Relationship between growth cone attraction and cone in Fig. 8E. No obvious differences in overall levels of F-actin protrusion were detected, but slight changes would not be distinguished using Our findings suggest that there is a link between an attractive cue this assay. unc-40-null mutations had no effect on VAB- and stimulation of protrusion, and between a repulsive cue and 10ABD::GFP distribution. These results suggest UNC-5 and UNC- inhibition of protrusion in the growth cone. When the repulsive 6 control the distribution of F-actin in the VD growth cone away receptor UNC-5 is removed in VDs, the result is an overly from the UNC-6/netrin source and towards the protrusive dorsal protrusive phenotype. When the attractive UNC-40/DCC receptor, edge of the growth cones. or its ligand UNC-6/netrin, are removed in HSNs, the result is reduced protrusiveness. DISCUSSION Similarly, when an activated version of the attractive receptor UNC-6/netrin and its receptors modulate extent of UNC-40/DCC is expressed in HSNs, it causes an increase in growth cone protrusion protrusiveness. However, when it is expressed in VDs containing We show here that in UNC-6-repelled VD neurons, which express UNC-5, it causes excess inhibition of protrusion, probably through both UNC-40/DCC and UNC-5, loss of unc-5 causes an increase the formation of repulsive protrusion-limiting UNC-5/UNC- in protrusive activity. We show that this is probably due to an 40/DCC heterodimers. increase in protrusion-stimulating activity of UNC-6/netrin and These findings suggest a broader theme in which the sensation UNC-40/DCC signaling in the absence of protrusion-limiting of an attractive cue causes stimulation of protrusion, whereas a UNC-5. In the HSNs, we also show that loss of UNC-6/netrin or repulsive cue causes inhibition of protrusion. These in vivo UNC-40/DCC causes a reduction in the extent of growth cone morphological results are also supported by previous in vitro protrusion, probably owing to a loss of attractive signaling. Our studies (Shekarabi and Kennedy, 2002; Shekarabi et al., 2005). results suggest that UNC-6/netrin mediates a balance of protrusive activity in the growth cone, with UNC-40 driving protrusion and UNC-6/netrin and its receptors affect polarity of UNC-5/UNC-40 inhibiting protrusion. This might set up a gradient protrusion of protrusive activity, which in the VD growth cone results in more Previous studies have shown that UNC-6/netrin is involved in protrusion dorsally and less ventrally (see Movie 1 in the establishment of initial protrusion polarity in the growth cone supplementary material). This mechanism might in part explain (Adler et al., 2006). We expand upon these findings and show that growth cone polarity and directed outgrowth during growth cone UNC-6/netrin, UNC-40/DCC and UNC-5 are all involved in
pathfinding. The fact that there is still a low level of dorsoventral controlling the polarity of protrusion in the growth cone. DEVELOPMENT Netrin and the growth cone RESEARCH ARTICLE 4441
We show here that UNC-6/netrin is required for appropriately polarizing the protrusions of VD and HSN growth cones. In unc-6 mutants, both VD and HSN growth cones lose filopodia and F- actin polarization. We also show that in VDs, UNC-5 is required for appropriate polarization of filopodia and F-actin, and that in the HSNs UNC-40/DCC is required for appropriate polarization of filopodia.
Guidance in response to UNC-6/netrin might involve a balance of protrusion and inhibition of protrusion We have shown that UNC-6/netrin signaling affects both the extent of protrusion and polarity of protrusion and F-actin accumulation. Possibly, polarity of protrusion is achieved by asymmetric inhibition and stimulation of protrusion across the growth cone (e.g. in VD growth cones, ventral protrusion is more significantly inhibited, resulting in net dorsal protrusion) (see Movie 1 in the supplementary material). Consistent with this idea, we found that the over-protrusive effects of unc-5 and unc-6(e78) in VD growth cones required functional UNC-40, indicating that even in growth cones repelled from UNC-6/netrin there is still a latent pro- protrusive response to UNC-6/netrin. This is in agreement with the observation that there was a decrease in filopodia length in unc-40 VDs. Possibly, growth cone polarity involves balancing stimulation versus inhibition of protrusion across the growth cone in response to UNC-6/netrin. Indeed, we found a slight but significant increase in the duration and length of filopodia in the dorsal region of the VD growth cone compared with the ventral region. Although this effect was small, the outcome on growth cone movement when integrated over time could be significant. An attractive model is that UNC-40 signaling alone generally stimulates protrusion in response to UNC-6/netrin, and that UNC-5 and UNC-40 signaling inhibits protrusion in response to UNC-6/netrin close to the ventral UNC-6/netrin source (see Fig. S1 in the supplementary material). Fig. 8. Netrin signaling mutants cause loss of growth cone F-actin Growth cone polarity and dorsal migration might depend upon polarity. (A-D)Representative images of VD growth cones with asymmetric distribution of protrusive activities. Although the cytoplasmic mCherry in red (a volumetric marker) and the VAB- mechanisms are unclear, such asymmetry might be achieved via 10ABD::GFP in green. Areas of overlap are yellow. Scale bar: 5m. (A,B)In differential receptor complex sensitivity to UNC-6, or by feedback wild type, VAB-10ABD::GFP was predominantly at the dorsal edge of the that consolidates asymmetry (e.g. receptor trafficking). Supporting growth cones (arrows). (C,D)Representative images of unc-6(ev400) VD growth cones with cytoplasmic mCherry and VAB-10ABD::GFP expression. the latter notion, localization of UNC-40 and the SLT-1/Slit Both growth cones have undergone guidance errors. In C, the growth receptor SAX-3 by the UNC-73/Trio GEF, MIG-2,/RhoG and cone first grew dorsally, then back ventrally, and is shown growing VAB-8 are important for receptor function (Levy-Strumpf and laterally. The growth cone in D is advancing laterally. Dorsal accumulation Culotti, 2007; Watari-Goshima et al., 2007). of VAB-10ABD::GFP is not observed in either growth cone. The growth Endpoint analysis of VD axons also indicates that pathfinding cone in C displayed a ventral accumulation, and the growth in D displayed might involve a balance of protrusion and inhibition of protrusion. no asymmetric accumulation. (E,F)Representative line plots of a wild-type unc-5 and unc-6(e78) mutants had more severe axon pathfinding VD growth cone (E) and an unc-6(ev400) mutant growth cone (F) with errors than a null unc-6 allele, but removing unc-40 in either one VAB-10ABD::GFP expression. The x-axis represents distance (in pixels) of these backgrounds reverted the phenotype severity to that of the from the dorsal edge of the growth cone of a line drawn vertically from the dorsal growth cone to the ventral growth cone (see inset). The y-axis unc-6 null. This indicates that excess protrusion may lead directly represents the relative pixel intensity of VAB-10ABD::GFP compared with to increased axon pathfinding defect severity. Our results also the volumetric mCherry marker (GFP/mCherry). Five lines were drawn for suggest an additional mechanism for the previously observed high the growth cone and the pixel intensity ratios were averaged. At these level of axon pathfinding defects at short distances from the UNC- exposures, the GFP and mCherry intensities were comparable, such that 6/netrin source in unc-5 mutants (MacNeil et al., 2009). These the ratios were close to 1. These graphs represent data from single growth authors postulated that UNC-5 homodimers mediated repulsion cones, but others show a similar pattern. Error bars represent s.e.m. of the near the UNC-6/netrin source, and that UNC-5/UNC-40 intensity ratios of the five line scans. (G)The average dorsal-to-ventral ratio heterodimers mediated repulsion further from the UNC-6/netrin of GFP/mCherry from multiple growth cones. This graph was generated source loss. Our results suggest that in unc-5 mutants, latent UNC- using line scans on multiple growth cones as described in E and F. Growth 40 mediated attraction to UNC-6/netrin steers the growth cone back cones were divided into dorsal and ventral halves of identical area, and the average intensity ratio of VAB-10ABD::GFP/mCherry was determined for toward the ventral source of UNC-6, resulting in severe axon each half. The dorsal ratios were divided by the ventral ratios to determine pathfinding defects. These two mechanisms are not mutually the relative dorsal enrichment of VAB-10ABD::GFP. A mean and s.e.m. exclusive and both might contribute to the severe axon pathfinding
from multiple growth cones was generated and plotted. n≥10; *P≤0.05. defects of unc-5 mutants compared with unc-40 and unc-6 mutants. DEVELOPMENT 4442 RESEARCH ARTICLE Development 138 (20)
UNC-6/netrin signaling controls growth cone Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. and Hedgecock, E. M. filopodial dynamics (1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9, 873-881. In contrast to involving balanced protrusion across the growth Jin, Y., Jorgensen, E., Hartwieg, E. and Horvitz, H. R. (1999). The cone, the extent of protrusion and the polarity of protrusion might Caenorhabditis elegans gene unc-25 encodes glutamic acid decarboxylase and is be regulated by separate mechanisms. Supporting this idea are our required for synaptic transmission but not synaptic development. J. Neurosci. 19, observations that in unc-5 and unc-6(e78) mutants, the length and 539-548. Knobel, K. M., Jorgensen, E. M. and Bastiani, M. J. (1999). Growth cones stall duration of VD growth cone filopodia are increased, including and collapse during axon outgrowth in Caenorhabditis elegans. 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