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-offunction 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.
INTRODUCTION
Developing axons are led by growth cones, dynamic actin-based
structures that sense and respond to extracellular cues, driving the
forward motion of the axon (Mortimer et al., 2008; Tessier-Lavigne
and Goodman, 1996). Growth cones consist of lammelipodial
protrusions of a branched actin meshwork and filopodial
protrusions of bundled actin filaments, which are both involved in
the proper growth of an axon to its target destination (Gallo and
Letourneau, 2004; Pak et al., 2008; Zhou and Cohan, 2004).
The laminin-like UNC-6/netrin guidance cue and its receptors
UNC-40/DCC (deleted in colorectal carcinoma) and UNC-5 have
been widely implicated in axon pathfinding in mouse, Drosophila,
C. elegans and other organisms (Chan et al., 1996; Hong et al.,
1999; Leonardo et al., 1997; Montell, 1999; Pan et al., 2010;
Shekarabi and Kennedy, 2002; Moore et al., 2007). In C. elegans,
UNC-6 is secreted by ventral cells and is necessary for guidance of
circumferential neurons (Ishii et al., 1992). The receptor UNC-40
is expressed in growth cones and mediates an attractive response
to UNC-6, driving the ventral guidance of circumferential axons,
while the receptor UNC-5 is expressed in dorsally guided growth
cones and mediates a repulsive response to UNC-6, driving the
dorsal guidance of circumferential axons (Hedgecock et al., 1990;
Hong et al., 1999).
Vertebrate DCC and UNC-5 both respond to netrin by the
formation of receptor dimers. In neurons expressing only DCC,
netrin binding causes the formation of an DCC homodimer, leading
Programs in Genetics and Molecular, Cellular, and Developmental Biology,
Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue,
Lawrence, KS 66045, USA.
*Author for correspondence ([email protected])
Accepted 1 August 2011
to an attractive response by the growth cone. In neurons expressing
both UNC-5 and DCC, netrin binding leads primarily to UNC-5
alone or UNC-5 and DCC signaling, both of which mediate a
repulsive response (Hong et al., 1999; MacNeil et al., 2009).
Whereas the role of UNC-6/netrin and its receptors have been
studied extensively in axon pathfinding endpoint analyses and in
vitro growth cone experiments, little is known about how they
affect the growth cone in vivo during growth cone outgrowth.
Previous studies have shown that UNC-6/netrin can affect the
polarity of growth cone protrusions in C. elegans (Adler et al.,
2006), and the morphology of the growth cone in neurons in vitro
(Shekarabi and Kennedy, 2002; Shekarabi et al., 2005). This led us
to speculate that UNC-6/netrin and its receptors may play an
important role in modulating both the extent and the polarity of
protrusion in C. elegans growth cones.
We endeavored to understand how UNC-6/netrin and its
receptors UNC-40/DCC and UNC-5 affect axon pathfinding,
growth cone morphology and dynamics in circumferential
neurons in C. elegans, using methods we have previously
developed that allow in vivo analysis of C. elegans growth
cones. We show here that UNC-6/netrin and its receptors UNC40/DCC and UNC-5 are involved in both polarity of growth
cone protrusiveness as well as in modulating the extent of
protrusion. Indeed, these molecules affected filopodial dynamics
independently of their polarity of protrusion, suggesting that
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
of protrusion within a growth cone, with UNC-6 driving
protrusion via UNC-40 and inhibiting protrusion via UNC-5.
This balance might establish a dorsal-ventral gradient of
protrusion across the growth cone, which in part might underlie
growth cone polarity and directed outgrowth.
DEVELOPMENT
KEY WORDS: Filopodia, Growth cone, Lamellipodia, Protrusion, UNC-6/netrin
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).
Briefly, animals harboring the juIs76 [unc-25::gfp] transgene were selected
16 hours post-hatching at 20°C and placed on a 2% agarose pad with a drop
of 10 mM muscimol (Sigma-Aldrich, St Louis, MO, USA) in M9 (Weinkove
et al., 2008), which was allowed to evaporate for 3-4 minutes before placing
coverslip on top of the animals on the pad. Growth cones were imaged with
a Qimaging Rolera mGi EMCCD camera using a very short exposure time
(~30 mseconds) on a Leica DMR microscope. Images were acquired at
intervals of 120 seconds, with total duration of time lapse ranging from 20
to 60 minutes. HSN growth cone time-lapse imaging was performed
identically, except that animals were selected at 19 hours post hatching.
Growth cone protrusions were scored as previously described (Norris et
al., 2009). Dynamic projections less than 0.5 m in width emanating from
the growth cone were scored as filopodia; protrusions greater than 0.5 m
were considered lamellipodial extensions; and protrusions that were static
were not considered filopodia. Filopodia were considered to have
disappeared at the point when there was no longer a visible a protrusion of
less than 0.5 m protruding from the membrane at the location where the
filopodium had once been. Filopodia length and growth cone area were
measured using ImageJ software. Filopodia length was determined by
drawing a straight line from the base where the filopodium originates on
the edge of the peripheral membrane to the tip of the filopodium. Polarity
of filopodia protrusion was determined using ImageJ and dividing the
growth cone into roughly four equally sized quadrants (dorsal-anterior,
dorsal-posterior, ventral-anterior and ventral-posterior) and determining
from which quadrant each filopodium emanated (see Fig. 7B for example).
Significance of difference was determined in all cases by a two-sided t-test
with unequal variance or a Fisher’s exact test.
Imaging of axon guidance defects
VD neurons were visualized with an unc-25::gfp (Jin et al., 1999) or unc25::mCherry transgene, expressed in all GABAergic neurons, including the
16 VDs. VD axons were considered defective if the axon failed to reach
the dorsal nerve cord or if it branched or turned at an angle greater than 45°
before reaching the dorsal nerve cord. To determine severity of defects, a
myo-3::cfp transgene was introduced to mark the ventral and dorsal muscle
quadrants. Using an unc-25::mCherry construct we could then determine
whether an the trajectory of an axon went awry before or after reaching the
dorsal edge of the ventral muscle quadrant.
HSNs were visualized with an unc-86::gfp transgene (Shen and
Bargmann, 2003), which is expressed in numerous neurons, including the
two HSNs. HSN axons were considered defective if the axon failed to
reach the ventral nerve cord, or if it branched or turned at an angle greater
than 45° before reaching the ventral nerve cord.
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: 5m. 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 2⫻ standard error of the proportion.
DEVELOPMENT
Experiments performed at 20°C using standard C. elegans techniques
(Brenner, 1974). These mutations and transgenics were used: X, unc6(ev400 and e78), lqIs170 [rgef-1::vab-10ABD::gfp]; I, unc-40(n324 and
e1430), unc-73(rh40); II, juIs76 [unc-25::gfp]; IV, kyIs179 [unc-86::gfp],
unc-5(e53 and e152); ?, lqIs128 [unc-25::myr::unc-40], lqIs129 [unc25::myr::unc-40], lqIs164 [myo-3::cfp], lqIs182 [unc-86::myr::gfp] and
lhIs6 [unc-25::mCherry]. Extrachromosomal arrays were attained by
injection into the germline, and then integrated into the genome via
standard techniques (Mello and Fire, 1995). Double mutants with unc-6,
unc-5 and unc-40 were confirmed by the uncoordinated movement
phenotype, axon pathfinding phenotype and PCR genotyping.
VAB-10ABD::GFP for F-actin visualization
F-actin was visualized by imaging growth cones expressing the F-actin
binding domain of spectraplakin VAB-10 fused to GFP, which has previously
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
localization of a cytoplasmic mCherry to control for nonspecific factors such
as variable growth cone size and shape. Asymmetric accumulation was
quantified with line scans through the growth cone on the dorsal-ventral axis
using NIH Image (see Fig. 8). Five line scans were made through each
growth cone, and the average pixel intensity of the green channel was
divided by the average intensity of the red channel to yield a relative intensity
ratio indicating the level of enrichment of the VAB-10ABD::GFP construct.
RESULTS
UNC-5, UNC-6 and UNC-40 control dorsal-ventral
axon pathfinding
In C. elegans, unc-6 and unc-40 mutants display guidance defects
in both ventrally directed and dorsally directed circumferential
axons, and unc-5 mutants display defective pathfinding in dorsally
directed circumferential axons (Hedgecock et al., 1990). We
confirmed and quantified defects in unc-5, unc-6 and unc-40
mutants in the dorsally directed VD motoneurons that are repelled
from UNC-6, and in the ventrally directed HSN axons that are
attracted to UNC-6 (Fig. 1). Consistent with previous results, unc5 and unc-6 mutants displayed a nearly complete failure of VD
axons to reach the dorsal nerve cord, whereas unc-40 mutants had
a weaker effect on VD axon pathfinding (Fig. 1A-D). By contrast,
unc-6 and unc-40 mutants had strong HSN axon guidance defects,
whereas unc-5 mutants did not (Fig. 1F-H). The unc-6(e78) allele,
which results in a cysteine to tyrosine substitution in domain V-3
that disrupts interaction with UNC-5 but not UNC-40/DCC (Lim
and Wadsworth, 2002), more strongly affected dorsal repulsive
growth than ventral attractive growth, as previously reported (Fig.
RESEARCH ARTICLE 4435
1A,F). These results are consistent with previous findings that
UNC-6/netrin and UNC-40/DCC, but not UNC-5, are required for
ventral migration (Hedgecock et al., 1990).
UNC-5 and UNC-6 inhibit growth cone protrusion
Although it is established that UNC-6/netrin signaling affects
circumferential axon guidance, the effects of these molecules on
the growth cone during outgrowth in vivo are less well understood.
We imaged the growth cones of the VD motoneurons that are
repelled from UNC-6/netrin over time as they extended. VD
growth cones were imaged in animals at 16 hours post-hatching at
a time when the VD growth cones have begun their commissural
migrations (see Materials and methods for imaging methods). We
measured growth cone area as well as the dynamic parameters of
filopodia initiation rate, filopodia duration and maximal filopodial
length (see Materials and methods for parameter measurement).
Wild-type VD growth cones were dynamic (see Movie 1 in the
supplementary material) and had an average area of 6.7 m2 (Fig.
2A). Wild-type growth cones also displayed multiple dynamic
filopodia that formed at an average rate of 0.26 per minute (one
initiation event every 3.8 minutes) (Fig. 2B,F). The average wildtype filopodium had a duration of 4.9 minutes (between the time it
was first noticeable to the time it was no longer apparent) (Fig. 2C)
and had an average maximal length of 0.96 m (Fig. 2D). These
parameters are similar to those previously observed for wild-type
VD growth cones (Norris et al., 2009).
To understand how axonal repulsion relates to growth cone
morphology during outgrowth, we analyzed VD growth cones in
unc-5 mutants. unc-5 caused an increase in growth cone
protrusiveness in VD growth cones (see Movie 2 in the
supplementary material). Growth cone area was significantly
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 inm2. (B)Filopodia
formation rate, in initiation events per minute.
(C)Duration of filopodia once formed, in minutes.
(D)Maximum filopodia length, inm. (E)Percentage of
filopodia greater than the wild-type average + 2 standard
deviations in length. Error bars represent 2⫻ 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: 5m.
DEVELOPMENT
Netrin and the growth cone
maximal filopodial length was also increased significantly (Fig.
2D). However, filopodial length appeared biphasic in unc-5
mutants, with many filopodia that were comparable with wild-type
length and some that grew very long (greater than two standard
deviations from average wild-type length) (Fig. 2E,G; see Movie 1
in the supplementary material). Some of these long filopodia were
observed to endure throughout the growth cone imaging period,
suggesting that they are very stable growth cone protrusions not
observed in wild type. These data indicate that UNC-5 is normally
required to limit the extent of protrusion of growth cones that are
repelled from UNC-6. unc-5 mutants did not affect the rate of
filopodia formation (Fig. 2B), suggesting that UNC-5 might affect
filopodial dynamics after a filopodium has been formed.
Surprisingly, the unc-6(ev400) null mutation had no significant
effect on any of the VD growth cone parameters we measured (Fig.
2A-E). However, the unc-6(e78) mutation that specifically disrupts
the repulsive, UNC-5-dependent role of UNC-6 (Lim and
Wadsworth, 2002), resembled unc-5 mutants in all parameters
measured (increased growth cone area, increased filopodial
duration and unusually long filopodia with no effect on filopodia
initiation rate) (Fig. 2A-E). That these effects are reduced in the
unc-6 null suggests that in unc-6(e78), increased growth cone
protrusiveness was due to a non-UNC-5 mediated role of UNC-6,
possibly interaction with UNC-40. Indeed, as we show below, loss
of UNC-40 suppressed the increased filopodial dynamics in unc-5
mutants and in unc-6(e78), indicating that UNC-40 is required for
increased growth cone protrusion in these mutants.
Constitutively-active MYR::UNC-40 inhibits VD
growth cone protrusion
Two putative null mutations in unc-40, e1430 and n324, had very
little effect on VD growth cone dynamics and morphology (Fig.
2A-E), with the only significant effect being a reduction in
Development 138 (20)
maximal filopodial length compared with wild type (Fig. 2D). This
is surprising given that UNC-40 is thought to cooperate with UNC5 in repulsive axon guidance.
To probe the role of UNC-40 in VD growth cone dynamics in
more detail, we constructed a constitutively active version of UNC40, based upon Gitai et al. (Gitai et al., 2003); by adding an Nterminal myristoylation signal to the cytoplasmic domain of UNC40. MYR::UNC-40 lacks the transmembrane domain and the
extracellular domains, which function to inhibit dimerization in the
absence of UNC-6/netrin. MYR::UNC-40 is targeted to membranes
by the myristoylation sequence and is thought to be constitutively
active even in the absence of UNC-6/netrin.
Expression of myr::unc-40 was driven in the VD neurons using
the unc-25 promoter. In a wild-type background, MYR::UNC-40
caused a marked decrease in VD growth cone protrusiveness (see
Movie 3 in the supplementary material). MYR::UNC-40 growth
cones were small [6.8 m2 in wild type compared with 4.6 m2 in
lqIs128[myr::unc-40]; P<0.01 (Fig. 2A)] with few filopodial
protrusions, and exhibited a ‘treadmilling’ behavior in which there
was some movement around the edges of the growth cone but very
little growth cone advance (see Movie 3 in the supplementary
material). MYR::UNC-40 growth cones exhibited a decrease in
filopodial formation rate (0.26 per minute in wild-type compared
with 0.16 per minute in lqIs128[myr::unc-40]; P<0.01) (Fig. 2B).
When filopodia did form, they had reduced duration (Fig. 2C) and
reduced maximal length (Fig. 2D). These data suggest that UNC-40
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
also affected filopodia initiation rate. Thus, UNC-40 might also be
involved in inhibition of filopodial initiation. Alternatively, filopodia
might form at a normal rate in myr::unc-40 but are inhibited before
they are visible in our imaging conditions, which might not be able
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 2⫻
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: 5m.
DEVELOPMENT
4436 RESEARCH ARTICLE
RESEARCH ARTICLE 4437
defects in growth cone morphology, myr::unc-40 caused defects in
VD and DD dorsal axon pathfinding, as determined by end-point
analysis (55% defective axons in lqIs128[myr::unc-40]) (Fig. 1A).
That loss of unc-5 and activation of myr::unc-40 had opposite effects
on growth cone protrusiveness indicate that these are bona fide
effects of the molecules on the growth cone and not due to a general
perturbation of growth cone function.
excess protrusion in unc-5 mutants was due in part to slow
advance. We think this is unlikely because MYR::UNC-40 growth
cones, which also displayed slow advance, had reduced protrusion;
and because unc-5 loss of function suppressed the inhibition of
protrusion of MYR::UNC-40 and restored protrusion to unc-5-like
levels, indicating that UNC-5 was directly required for inhibiting
protrusion.
Constitutively active MYR::UNC-40 requires UNC-5
to inhibit VD protrusion
We next tested whether the MYR::UNC-40-mediated reduction in
growth cone protrusion was dependent on the presence of other
netrin signaling components. We found that the MYR::UNC-40
phenotype was not dependent on UNC-6, as unc-6(ev400); myr::unc40 double mutant VD growth cones resembled myr::unc-40 alone
(Fig. 3A-D). This result is consistent with the idea that MYR::UNC40 is an UNC-6-independent, constitutively active molecule.
MYR::UNC-40 was also not dependent on endogenous UNC-40
(Fig. 3A-D). However, MYR::UNC-40 inhibition of protrusion was
dependent upon UNC-5, as unc-5(e53) suppressed the effects of
myr::unc-40 (Fig. 3A-F, see Movie 4 in the supplementary material).
unc-5(e53) restored growth cone area, filopodia initiation rate,
duration and maximal length (Fig. 3A-D). unc-5(e53); myr::unc-40
had a protrusion comparable with or greater than that of wild type,
and in fact resembled the excessive protrusion seen in unc-5 mutants
alone (e.g. filopodial duration was increased in unc-5(e53);
lqIs128[myr::unc-40] compared with wild type; Fig. 3C).
These results suggest that MYR::UNC-40 requires UNC-5 to
inhibit growth cone protrusion, consistent with previous results
suggesting that an UNC-40/UNC-5 heterodimer mediates growth
away from UNC-6/netrin. These results also suggest that formation
of a putative MYR::UNC-40/UNC-5 heterodimer is independent
of UNC-6/netrin, as would be expected of a ligand-independent
constitutively active molecule.
Previous studies indicated that stationary or slow-moving growth
cones often excess protrusion (Knobel et al., 1999), and we found
that unc-5 mutant growth cones exhibited slow advance. Possibly,
VD growth cone over-protrusive phenotypes of
unc-5 and unc-6(e78) are suppressed by unc-40
mutation
unc-5 null and unc-6(e78) mutants but not unc-6(ev400) null
mutants displayed increased growth cone protrusion, suggesting a
role of UNC-6 in stimulating VD growth cone protrusion in the
absence of UNC-5. We speculated that this might involve UNC-40.
We constructed unc-5; unc-40 double mutants and found that they
did not exhibit the over-protrusive phenotypes seen in the unc-5
single mutants (Fig. 4): growth cone size, filopodial duration and
filopodia length (Fig. 4A-C) all returned to near-wild-type levels
in the double mutants using two distinct null alleles of unc-40.
Similarly, unc-40(n324) was required for the over-protrusive
growth cones of unc-6(e78) mutants, as unc-6(e78); unc-40(n324)
double mutants had near wild-type levels of protrusion (Fig. 4AC). These results suggest that UNC-40 and UNC-6 have roles in
stimulating VD growth cone protrusion in addition to roles in
inhibition of protrusion along with UNC-5, as demonstrated by the
MYR::UNC-40 results above. The above findings might also
explain why unc-6-null mutants and unc-40 mutants display less
severe growth cone protrusion defects than unc-5 mutants (i.e.
UNC-6 and UNC-40 are required both to stimulate and to inhibit
protrusion, and when they are gone an equilibrium between the two
is maintained).
This balance of protrusion in the growth cone leads to the idea
that protrusion might be asymetrically affected across the growth
cone, which might explain in part growth cone polarity and
directed outgrowth. Indeed, in wild-type VD growth cones,
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 2⫻ 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: 5m.
DEVELOPMENT
Netrin and the growth cone
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).
Netrin signaling component mutants modulate
extent of protrusion of HSN growth cones
To study the roles of UNC-6 and UNC-40 in growth cones that are
attracted to UNC-6/netrin, we turned to the HSN neurons. The
bilateral HSN cell bodies reside laterally just behind the
hermaphrodite vulva in the midsection of the animal. The HSN
axons extend ventrally to the ventral nerve cord and then anteriorly
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
HSN axons often extend laterally instead of ventrally. The HSN
displays a prominent growth cone at 18-19 hours post-hatching in
the late L1 stage (see Movie 5 in the supplementary material). At this
time, the growth cone resembles a thickened protrusion from the
ventral region of the cell body with multiple filopodial protrusions,
as previously reported (Adler et al., 2006). Over the next 12 hours
into the mid L4 larval stage, the HSN growth cone remains dynamic
and migrates ventrally to the ventral nerve cord. We analyzed HSN
growth cones in time lapse at 19 hours post-hatching. At this time
the wild-type HSN growth cone is on average 11.2 m2 in size, with
filopodia that form at an average rate of one every 4.4 minutes and
endure for 5.2 minutes, with a maximal filopodial length of 0.9 m
(Fig. 6 and see Movie 5 in the supplementary material). The HSN
growth cone is larger, but all dynamic filopodial characteristics are
similar to those of the VD growth cones.
unc-40- and unc-6-null mutant HSN growth cones exhibited a
decrease in protrusiveness (see Movie 6 in the supplementary
material), with a decrease in growth cone, filopodial duration and
filopodia length (Fig. 6A-D). unc-6(ev400)-null mutants but not unc40 mutants also exhibited a decrease in filopodia formation rate (Fig.
6B). These results indicate that UNC-6/netrin and UNC-40/DCC are
Fig. 5. Severity of unc-5(e53) axon pathfinding defects is
mitigated by loss of unc-40. (A)Percentage of VD axons that have
pathfinding errors by the time they reach the dorsal edge of the ventral
muscle quadrant. Error bars represent 2⫻ standard error of the mean;
asterisks indicate a significant difference between wild type and the
mutant (P<0.01); + indicates a significant difference between unc5(e53) alone and the unc-5(e53); unc-40(n324) double mutant (P<0.01)
determined by two-sided t-test with unequal variance.
(B,C)Representative images showing VD axons (arrows) with mCherry
expression (red) after their complete outgrowth in L4 animals. Ventral
and dorsal muscle quadrants with CFP expression are blue. In unc5(e53) most axons do not advance beyond the ventral muscle
quadrants, whereas in unc-5(e53); unc-40(n324), many axons extend
past the ventral quadrants. Scale bar: 5m.
required for robust HSN growth cone protrusion. As expected, the
unc-6(e78) mutation, which specifically attenuates UNC-5-mediated
effects of UNC-6, had no effect on HSN growth cones (Fig. 6).
We drove the expression of MYR::UNC-40 in the HSNs using
the unc-86 promoter. In contrast to inhibiting protrusion in the
VDs, MYR::UNC-40 caused an increase in protrusiveness in the
HSNs (see Movie 7 in the supplementary material), with an
increase in growth cone size, filopodia duration and length (Fig. 6).
This is the opposite effect seen with MYR::UNC-40 in the VDs,
and suggests that in axons that do not express UNC-5, the
MYR::UNC-40 molecules form an attractive complex that
stimulates growth cone protrusion.
Considered with the results in the VD growth cones, these data
suggest that UNC-6/netrin and it receptors UNC-40/DCC and UNC5 control the extent of growth cone protrusion, including growth cone
size and the maximal length and duration of growth cone filopodia.
Inhibition versus stimulation of protrusion were correlated with
repulsion versus attraction in response to UNC-6/netrin. However, in
the VD growth cones, unc-6 mutants had only weak effects on growth
cone protrusiveness but very strong VD axon guidance defects,
indicating that regulation of growth cone protrusion is not the only
role of UNC-6/netrin in growth cones during axon pathfinding.
DEVELOPMENT
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 UNC5 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. unc40(n324); unc-5(e53) double mutants were less severe than unc5(e53) alone, and had guidance defects of comparable severity to
unc-6(ev400) alone. These data indicate that in unc-5 mutant VD
growth cones, UNC-40/DCC may be mediating an attractive
response to UNC-6/netrin, causing these axons to become attracted
to, rather than repelled from, UNC-6/netrin, and thus misguided
sooner than in the complete absence of UNC-6/netrin.
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 2⫻
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 unc40(n324) HSN growth cone, which often protrudes
anteriorly and laterally rather than ventrally from the cell
body. Arrowheads indicate growth cones. Scale bar: 5m.
from the anterior of the cell body. Loss of UNC-5 did not affect
dorsal-ventral polarity, which is consistent with the observation
that UNC-5 is not expressed in the HSN (Fig. 7E,H). However,
loss of UNC-40/DCC or UNC-6/netrin abolished the ventral
polarity of filopodia extension (Fig. 7E,G,H), and the growth
cone often extended anterolaterally instead of ventrally (Fig.
7G). These results are consistent with previous results showing
that UNC-6 localizes UNC-40 to the ventral surface of the HSN
and, in an unc-6 mutant, UNC-40 localizes uniformly around the
cell body and protrusions extend from around the cell body (Xu
et al., 2009). These results indicate that UNC-5, UNC-6 and
UNC-40 control the polarity of filopodial protrusion from
growth cones that are repelled (VDs) or attracted (HSNs) to
UNC-6/netrin.
UNC-5 and UNC-6 control polarity of growth cone
filamentous actin
Growth cone protrusion, including the lamellipodial growth cone
body and filopodial extensions, involves dynamic regulation of the
actin cytoskeleton (Quinn and Wadsworth, 2008). In the VDs and
the PQR dendrite growth cones, the actin regulators Arp2/3, UNC115/abLIM and UNC-34/enabled are required for robust filopodial
extensions (Norris et al., 2009). We assayed filamentous actin (Factin) in the VD growth cones, using a construct in which the Factin binding domain of the spectraplakin VAB-10 is fused to GFP
that has been used to monitor F-actin in other cells in living C.
elegans (Bosher et al., 2003; Patel et al., 2008). In wild-type VD
growth cones, this VAB-10ABD::GFP construct localized
preferentially to the dorsal leading edge of the lamellipodia and in
filopodial protrusions (Fig. 8A,B,E). On average, VAB10ABD::GFP showed 1.22-fold more accumulation in the dorsal
half of growth cones compared with the ventral halves (Fig. 8G).
In unc-6(ev400)-null mutants and unc-5(e53)-null mutants, VAB10ABD::GFP no longer showed a preferential dorsal leading edge
accumulation (Fig. 8C,D,G). Rather, VAB-10ABD::GFP
DEVELOPMENT
UNC-5, UNC-6 and UNC-40 affect polarity of
growth cone filopodia protrusion
Wild-type VD and HSN growth cones display highly polarized
filopodial protrusion: in VD growth cones, most filopodia
protrude dorsally away from UNC-6 (Fig. 7A,B,D); and in HSN
growth cones, most filopodia protrude ventrally toward UNC-6
(Fig. 7E,F,H). We next determined whether orientation of
filopodial protrusion was altered in unc-5, unc-6 and unc-40
mutants. Previous studies indicated that UNC-6 was required for
polarized distribution of UNC-40 and MIG-10/lamellipodin on
the ventral region of the HSN neuron and that unc-6 and unc-40
mutant HSN neurons displayed a loss of polarized ventral
protrusion (Adler et al., 2006; Quinn et al., 2006; Quinn et al.,
2008; Xu et al., 2009). To quantify polarity of filopodial
protrusion, we divided the growth cones into four quadrants
along the AP and DV axes, based upon the orientation of the
animal, regardless of the direction that the growth cone was
navigating (some VD growth cones in mutants were misguided
and were migrating laterally or even ventrally; and many HSN
growth cones protruded laterally rather than ventrally). In wild
type, VD neurons growing along naked epidermis, the majority
of filopodia (70%) extended from the dorsal half of the growth
cone (Fig. 7A,B,D). unc-5- and unc-6-null mutations abolished
this polarity such that filopodia protruded nearly equally in the
dorsal and ventral directions (Fig. 7A,C,D). During the stage at
which we imaged VD growth cones, unc-40-null mutations had
no significant effect on dorsal ventral polarity of VD growth
cone filopodial protrusion, nor did MYR::UNC-40. Anteriorposterior VD filopodial protrusion was not significantly different
in wild-type or any mutant (Fig. 7D).
In wild-type HSN growth cones, the majority of filopodia
extended from the ventral half of the growth cone, towards their
target in the VNC (Fig. 7E,F,H). In HSN growth cones of all
genotypes, there was a bias toward anterior versus posterior
protrusion, as the HSN growth cone generally extends ventrally
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 unc5(e53) growth cones, with a line drawing of the growth
cone and filopodial perimeter on the right. Scale bar: 5m
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 unc40(n324) growth cones, with arrows marking growth cone
protrusions. (H)Relative filopodial protrusion from each
quadrant of HSN growth cones as described in D.
DISCUSSION
UNC-6/netrin and its receptors modulate extent of
growth cone protrusion
We show here that in UNC-6-repelled VD neurons, which express
both UNC-40/DCC and UNC-5, loss of unc-5 causes an increase
in protrusive activity. We show that this is probably due to an
increase in protrusion-stimulating activity of UNC-6/netrin and
UNC-40/DCC signaling in the absence of protrusion-limiting
UNC-5. In the HSNs, we also show that loss of UNC-6/netrin or
UNC-40/DCC causes a reduction in the extent of growth cone
protrusion, probably owing to a loss of attractive signaling. Our
results suggest that UNC-6/netrin mediates a balance of protrusive
activity in the growth cone, with UNC-40 driving protrusion and
UNC-5/UNC-40 inhibiting protrusion. This might set up a gradient
of protrusive activity, which in the VD growth cone results in more
protrusion dorsally and less ventrally (see Movie 1 in the
supplementary material). This mechanism might in part explain
growth cone polarity and directed outgrowth during growth cone
pathfinding. The fact that there is still a low level of dorsoventral
guidance in unc-6 mutants suggests that other guidance signals,
such as UNC-129 or SLT-1 may still be affecting the migration of
VD and HSN growth cones (e.g. SLT-1 preventing HSN axons
from migrating dorsally).
Relationship between growth cone attraction and
protrusion
Our findings suggest that there is a link between an attractive cue
and stimulation of protrusion, and between a repulsive cue and
inhibition of protrusion in the growth cone. When the repulsive
receptor UNC-5 is removed in VDs, the result is an overly
protrusive phenotype. When the attractive UNC-40/DCC receptor,
or its ligand UNC-6/netrin, are removed in HSNs, the result is
reduced protrusiveness.
Similarly, when an activated version of the attractive receptor
UNC-40/DCC is expressed in HSNs, it causes an increase in
protrusiveness. However, when it is expressed in VDs containing
UNC-5, it causes excess inhibition of protrusion, probably through
the formation of repulsive protrusion-limiting UNC-5/UNC40/DCC heterodimers.
These findings suggest a broader theme in which the sensation
of an attractive cue causes stimulation of protrusion, whereas a
repulsive cue causes inhibition of protrusion. These in vivo
morphological results are also supported by previous in vitro
studies (Shekarabi and Kennedy, 2002; Shekarabi et al., 2005).
UNC-6/netrin and its receptors affect polarity of
protrusion
Previous studies have shown that UNC-6/netrin is involved in
establishment of initial protrusion polarity in the growth cone
(Adler et al., 2006). We expand upon these findings and show that
UNC-6/netrin, UNC-40/DCC and UNC-5 are all involved in
controlling the polarity of protrusion in the growth cone.
DEVELOPMENT
distribution was randomized across the growth cone (Fig. 8E) and
in some growth cones showed a ventral bias (Fig. 8C). In unc6(ev400), F-actin was often seen to accumulate at both the dorsal
and ventral margins of the growth cone (Fig. 8F). F-actin
accumulated in filopodial protrusions at the margin, so this might
represent randomized filopodial protrusion, as seen in the growth
cone in Fig. 8E. No obvious differences in overall levels of F-actin
were detected, but slight changes would not be distinguished using
this assay. unc-40-null mutations had no effect on VAB10ABD::GFP distribution. These results suggest UNC-5 and UNC6 control the distribution of F-actin in the VD growth cone away
from the UNC-6/netrin source and towards the protrusive dorsal
edge of the growth cones.
Netrin and the growth cone
RESEARCH ARTICLE 4441
Fig. 8. Netrin signaling mutants cause loss of growth cone F-actin
polarity. (A-D)Representative images of VD growth cones with
cytoplasmic mCherry in red (a volumetric marker) and the VAB10ABD::GFP in green. Areas of overlap are yellow. Scale bar: 5m. (A,B)In
wild type, VAB-10ABD::GFP was predominantly at the dorsal edge of the
growth cones (arrows). (C,D)Representative images of unc-6(ev400) VD
growth cones with cytoplasmic mCherry and VAB-10ABD::GFP expression.
Both growth cones have undergone guidance errors. In C, the growth
cone first grew dorsally, then back ventrally, and is shown growing
laterally. The growth cone in D is advancing laterally. Dorsal accumulation
of VAB-10ABD::GFP is not observed in either growth cone. The growth
cone in C displayed a ventral accumulation, and the growth in D displayed
no asymmetric accumulation. (E,F)Representative line plots of a wild-type
VD growth cone (E) and an unc-6(ev400) mutant growth cone (F) with
VAB-10ABD::GFP expression. The x-axis represents distance (in pixels)
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
represents the relative pixel intensity of VAB-10ABD::GFP compared with
the volumetric mCherry marker (GFP/mCherry). Five lines were drawn for
the growth cone and the pixel intensity ratios were averaged. At these
exposures, the GFP and mCherry intensities were comparable, such that
the ratios were close to 1. These graphs represent data from single growth
cones, but others show a similar pattern. Error bars represent s.e.m. of the
intensity ratios of the five line scans. (G)The average dorsal-to-ventral ratio
of GFP/mCherry from multiple growth cones. This graph was generated
using line scans on multiple growth cones as described in E and F. Growth
cones were divided into dorsal and ventral halves of identical area, and the
average intensity ratio of VAB-10ABD::GFP/mCherry was determined for
each half. The dorsal ratios were divided by the ventral ratios to determine
the relative dorsal enrichment of VAB-10ABD::GFP. A mean and s.e.m.
from multiple growth cones was generated and plotted. n≥10; *P≤0.05.
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 proprotrusive 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).
Growth cone polarity and dorsal migration might depend upon
asymmetric distribution of protrusive activities. Although the
mechanisms are unclear, such asymmetry might be achieved via
differential receptor complex sensitivity to UNC-6, or by feedback
that consolidates asymmetry (e.g. receptor trafficking). Supporting
the latter notion, localization of UNC-40 and the SLT-1/Slit
receptor SAX-3 by the UNC-73/Trio GEF, MIG-2,/RhoG and
VAB-8 are important for receptor function (Levy-Strumpf and
Culotti, 2007; Watari-Goshima et al., 2007).
Endpoint analysis of VD axons also indicates that pathfinding
might involve a balance of protrusion and inhibition of protrusion.
unc-5 and unc-6(e78) mutants had more severe axon pathfinding
errors than a null unc-6 allele, but removing unc-40 in either one
of these backgrounds reverted the phenotype severity to that of the
unc-6 null. This indicates that excess protrusion may lead directly
to increased axon pathfinding defect severity. Our results also
suggest an additional mechanism for the previously observed high
level of axon pathfinding defects at short distances from the UNC6/netrin source in unc-5 mutants (MacNeil et al., 2009). These
authors postulated that UNC-5 homodimers mediated repulsion
near the UNC-6/netrin source, and that UNC-5/UNC-40
heterodimers mediated repulsion further from the UNC-6/netrin
source loss. Our results suggest that in unc-5 mutants, latent UNC40 mediated attraction to UNC-6/netrin steers the growth cone back
toward the ventral source of UNC-6, resulting in severe axon
pathfinding defects. These two mechanisms are not mutually
exclusive and both might contribute to the severe axon pathfinding
defects of unc-5 mutants compared with unc-40 and unc-6 mutants.
DEVELOPMENT
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 Factin 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.
UNC-6/netrin signaling controls growth cone
filopodial dynamics
In contrast to involving balanced protrusion across the growth
cone, the extent of protrusion and the polarity of protrusion might
be regulated by separate mechanisms. Supporting this idea are our
observations that in unc-5 and unc-6(e78) mutants, the length and
duration of VD growth cone filopodia are increased, including
dorsally directed filopodia, indicating an effect on filopodial
protrusion independent of their initiation or polarity. Furthermore,
MYR::UNC-40 activity in the HSN affects extent of protrusion but
has little effect on polarity of protrusion. Indeed, some filopodia in
unc-5 mutants grow very long and persist throughout the time of
imaging. Possibly, these persistent filopodia are the basis of the
ectopic axon branches observed in endpoint analysis of unc-5 and
unc-6 mutants, and that require functional UNC-40.
In summary, our results suggest UNC-6/netrin controls a balance
of protrusion within a growth cone, with UNC-40 driving
protrusion and UNC-5/UNC-40 inhibiting protrusion. Our results
also suggest that these forces might be asymmetric in the growth
cone, resulting in growth cone polarity and directed growth cone
protrusion and advance.
Acknowledgements
The authors thank members of the Lundquist and Ackley labs for helpful
discussion, S. Hamdeh for creation of numerous strains, E. Struckhoff for
technical assistance, M. Tourtellot for the Spoke12 program, and B. Ackley
(Lawrence, KS, USA), Y. Jin (San Diego, CA, USA), A. Chisholm (San Diego, CA,
USA), and M. Soto (Piscataway, NJ, USA) for reagents. Some nematode strains
used in this work were provided by the Caenorhabditis Genetics Center, which
is funded by the NIH National Center for Research Resources (NCRR).
Funding
This work was supported by NIH grant NS40945 to E.A.L. and by NIH grant
P20 RR016475 from the INBRE/IDEA Program of the National Center for
Research Resources (J. Hunt is the P.I.). Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.068841/-/DC1
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