© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 136-147 doi:10.1242/dev.095968
The second messengers cAMP and cGMP modulate attraction and
repulsion mediated by neuronal guidance cues. We find that the
Drosophila receptor guanylyl cyclase Gyc76C genetically interacts
with Semaphorin 1a (Sema-1a) and physically associates with the
Sema-1a receptor plexin A (PlexA). PlexA regulates Gyc76C catalytic
activity in vitro, and each distinct Gyc76C protein domain is crucial
for regulating Gyc76C activity in vitro and motor axon guidance in
vivo. The cytosolic protein dGIPC interacts with Gyc76C and
facilitates Sema-1a-PlexA/Gyc76C-mediated motor axon guidance.
These findings provide an in vivo link between semaphorin-mediated
repulsive axon guidance and alteration of intracellular neuronal cGMP
KEY WORDS: Receptor guanylyl cyclase, Gyc76C, cGMP,
Semaphorin-1a, Plexin A, dGIPC, Axon guidance
Both membrane-associated and secreted neuronal guidance cues can
attract or repel axons and dendrites during neural development, and
several families of guidance cues and receptors perform these
functions (Dickson, 2002; Kolodkin and Tessier-Lavigne, 2011).
Modulation of guidance cue activities through intracellular signaling
components determines how extrinsic factors are interpreted by
extending neuronal processes during development (Bashaw and
Klein, 2010). For example, growth cone turning experiments in vitro
demonstrate that attraction mediated by the guidance cue netrin-1
can be converted to repulsion by lowering intracellular cAMP (Ming
et al., 1997), whereas repulsion mediated by the guidance cue
Semaphorin 3A (Sema-3A) can be converted to attraction by
increasing intracellular cGMP (Song et al., 1998). Elevated cAMP
in cultured DRG neurons neutralizes Sema-3A growth cone
collapse, whereas elevated cGMP potentiates it (Dontchev and
Letourneau, 2002). The ratio of cAMP to cGMP can determine the
sign of a growth cone steering response (Nishiyama et al., 2003),
and Sema-3A induces cGMP production in neuronal growth cones,
activating of cGMP-gated calcium channels (CNGCs), Ca2+influx
and repulsion (Togashi et al., 2008). cAMP and cGMP regulate
kinases and phosphodiesterases to direct formation of axons or
dendrites in cultured hippocampal neurons (Shelly et al., 2010).
Therefore, coordination of cAMP and cGMP signaling regulates
cellular responses to different stimuli in the neurons.
Guanylyl cyclases (GCs) include soluble and transmembrane
proteins that catalyze the conversion of GTP to cGMP, and they
The Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical
Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205,
*Author for correspondence (email@example.com)
Received 26 February 2013; Accepted 26 September 2013
regulate a wide range of diverse cellular and physiological processes
(Davies, 2006), including axonal and dendritic guidance (Polleux et
al., 2000; Seidel and Bicker, 2000; Gibbs et al., 2001; Nishiyama et
al., 2003). The mammalian receptor guanylyl cyclase GC-B and
cGMP-dependent kinase I (cGKI) are essential for proper sensory
axon afferent guidance into the CNS, and C-type natriuretic peptide
is the GC-B ligand that is crucial for murine sensory axon
branching, axon outgrowth and axon attraction (Schmidt et al., 2009;
Zhao and Ma, 2009). Yet, how GCs are linked to axon guidance
signaling to alter intracellular cGMP levels and modulate growth
cone responses in vivo is unclear.
The Drosophila transmembrane semaphorin Sema-1a binds to the
plexin A (PlexA) receptor to mediate axon-axon repulsion and to
control axonal fasciculation in embryonic central and peripheral
nervous systems (CNS and PNS) (Winberg et al., 1998b; Yu et al.,
1998). The Drosophila receptor GC Gyc76C is required in
motoneurons for Sema-1a-PlexA-mediated axon guidance and is
dependent on the integrity of the Gyc76C catalytic cyclase domain
(Ayoob et al., 2004). Here, we investigate connections between
Gyc76C and Sema-1a-PlexA-mediated axon guidance. Our findings
support the theory that Gyc76C-generated cGMP within neuronal
growth cones facilitates axonal repulsion mediated by Sema-1a and
PlexA, allowing for the establishment of Drosophila embryonic
Gyc76C suppresses Sema 1a-mediated motor axon
Gyc76C mutations act as dominant enhancers of a Sema-1a-dependent
gain-of-function phenotype that affects CNS commissural axon
midline crossing in Drosophila embryos (embryos with this genotype
are referred to as ‘PUP’ for the genetic elements in this background)
(Ayoob et al., 2004). Altering Gyc76C gene dose modifies a PlexA-
dependent gain-of-function phenotype in CNS longitudinal connective
axons. Further, Gyc76C mutant embryos exhibit motor axon guidance
defects similar to Sema-1a mutant embryos, and Gyc76C genetically
interacts with Sema-1a and PlexA (Ayoob et al., 2004). These data
suggest Gyc76C functions in Sema-1a-PlexA-mediated motor axon
guidance. However, the PUP phenotypes are observed in a Sema-1a-
null genetic background in which Sema-1a is ectopically expressed on
CNS midline glia. However, the Gyc76C gain- and loss-of-function
phenotypes observed previously in motor axons (Ayoob et al., 2004)
do not allow for unequivocal assessments of responses to Sema-1a in
trans independent of roles Sema-1a and Gyc76C might play in axon-
axon interactions. Therefore, we employed a different Sema-1a gain-
of-function paradigm to investigate Gyc76C-mediated repulsive
signaling in motor axons in response to Sema-1a ligand presented in
Sema-1a is enriched in Drosophila embryonic neurons and
mediates axonal repulsion, ensuring proper axon pathfinding
(Winberg et al., 1998b; Yu et al., 1998; Ayoob et al., 2004; Cho et
Function of the Drosophila receptor guanylyl cyclase Gyc76C in
PlexA-mediated motor axon guidance
Kayam Chak and Alex L. Kolodkin*
RESEARCH ARTICLE Development (2014) doi:10.1242/dev.095968
al., 2012; Jeong et al., 2012). During neural development, motor
axons exit the CNS in two large bundles that include multiple motor
axons which then segregate into smaller motor nerves: the
intersegmental nerves (ISNs: ISNb and ISNd) and the segmental
nerves (SNs: SNa and SNc) (Landgraf et al., 1997). The fasciclin II
antibody 1D4 labels all motor axons, revealing stereotypic
embryonic neuromuscular connectivity (Grenningloh et al., 1991;
Van Vactor et al., 1993). ISNb axons defasciculate from the main
ISN bundle and navigate along ventral longitudinal muscles,
including muscles 6, 7, 12 and 13, to innervate appropriate targets
(Van Vactor et al., 1993; Landgraf et al., 1997). At each choice
point, the ISNb bundle extends nascent projections anteriorly and
posteriorly between muscles, establishing initial presynaptic contacts
with target muscles (i.e. RP3 and RP5 motor axons leave the ISNb,
then innervate muscles 6 and 7, and muscles 12 and 13,
respectively) (Fig. 1A).
We ectopically expressed Sema-1a in all embryonic muscles using
the Mef2-Gal4 driver (Ranganayakulu et al., 1996). Since Sema-1a
is a motor axon repellent (Winberg et al., 1998a; Winberg et al.,
1998b; Yu et al., 1998; Yu et al., 2000), we anticipated that Sema-1a-
expressing muscles would influence ISNb axons (Fig. 1A, red
circles). Removal of a signaling component involved in Sema-1a-
mediated axon guidance should suppress, or enhance, gain-of-
function phenotypes resulting from muscle-derived Sema-1a. We
confirmed muscle expression of Sema-1a in both UAS:Sema-1a/+,
Mef2-Gal4/+ embryos (Fig. 1B) and UAS:Sema-1a/+, Mef2-Gal4/+;
Gyc76Cex173/+ embryos (K.C. and A.L.K., unpublished) at embryonic
stage 16 by immunohistochemistry with anti-Sema-1a (Yu et al.,
1998). We observed a range of ISNb stalling and axon pathfinding
defects categorized into five distinct phenotypes (see Table 1): (1)
aberrant projection onto muscle 12 (M12); (2) stalling between
muscles 12 and 13 with no accompanying arborization (M12/13); (3)
stalling at muscle 13, or between muscles 13 and 6 (M13, M13/6);
(4) no presynaptic arborizations between muscles 6 and 7 (M6/7);
and (5) distinct ISNb defects, including bypasses (BPs) and also
axon-positioning defects (PDs). UAS:Sema-1a/+, Mef2-Gal4/+
embryos exhibited ISNb defects in 54.0% of hemisegments; the
majority of these defects were ISNb axon bundles stalled between
muscles 12 and 13 (25.8%) (Table 1). Axon pathfinding defects,
including those at M13 and M13/6 or at M6/7, were observed in
7.0% and 9.4% of hemisegments, respectively (Table 1). A
significant number of ISNb BPs (8.0% of hemisegments) and PDs
(8.4% of hemisegments) were observed in this Sema-1a gain-of-
function paradigm (Fig. 1C, open arrowheads). ISNb BP events,
including fusion and parallel bypasses, indicate a failure of the ISNb
to innervate the entire ventral muscle field, resulting in ISNb dorsal
extension along, or directly adjacent to, the ISN (Lin et al., 1994;
Desai et al., 1996; Yu et al., 1998; Wills et al., 1999). The observed
PDs are distinct ISNb pathfinding defects where the ISNb bundle
does not deviate from the ISN bundle but RP3 or RP5 neurons still
innervate target ventral muscles (Fig. 1C: open arrowhead, BP;
asterisk, muscle innervation). Therefore, Sema-1a presented in trans
on muscles acts as a motor axon repellent.
Removing one copy of Gyc76C produced significant reductions
in total ISNb defects (Fig. 1C′, open arrows), from 54.0% to 29.5%
(Fig. 1D; Table 1; P<0.005). Furthermore, both BPs and PDs were
suppressed: from 8% to 1.2%, and from 8.4% to 3.2%, respectively,
in UAS:Sema-1a/+, Mef2-Gal/+; Gyc76Cex173/+ embryos. We
observed similar suppression of these same phenotypes when one
copy of PlexA was removed in this gain-of-function paradigm
(UAS:Sema-1a/+, Mef2-Gal4/+;; PlexADf(4)C3/+) (Fig. 1D and
Table 1). Furthermore, UAS:Sema-1a/+; Gyc76Cex173/+ embryos
Fig. 1. Gyc76C suppresses Sema-1a-
mediated repulsion of motor axons in the
peripheral nervous system. (A) Schematic
diagram of wild-type (left) and Sema-1a
muscle gain-of-function (right) Drosophila
embryonic hemisegments showing ISNb
phenotypes (red circles). Anterior is
leftwards; dorsal is upwards. (B) Filleted
stage 16 Drosophila embryo harboring
UAS:Sema1a/+, Mef2-GAL4/+ transgenes
stained with the anti-fasciclin II (1D4, red)
and anti-Sema-1a (green). Scale bar: 10
μm. (C,C′) Three hemisegments of late
stage 16 embryos stained with 1D4. (C) In
UAS:Sema-1a/+, Mef2-Gal4/+ embryo,
ISNb motor axons often fail to reach their
ventral muscle targets (black arrows) or
exhibit pathfinding defects (open
arrowheads), including ISNb bypasses
(BPs) and positioning defects (PDs,
asterisk; see text). (C′) In UAS:Sema1a/+,
Mef2-GAL4/+; Gyc76Cex173/+ embryos,
Sema-1a gain-of-function ISNb stalling
phenotypes are greatly suppressed (open
arrows). Scale bar: 5 μm. (D) Quantification
of total ISNb pathfinding defects, PBs and
PDs following Sema-1a overexpression in
muscles. Z-test for two proportions defines
significant differences between genotypes;
*P<0.005 (see Table 1 for n values).
RESEARCH ARTICLE Development (2014) doi:10.1242/dev.095968
(1:500; Jackson ImmunoResearch), and Alexa488-conjugated goat anti-
mouse IgG, Alex546-conjugated goat anti-rabbit IgG and Alex647-
conjugated goat anti-rabbit IgG (all 1:500; Molecular Probes) were the
Live cell-surface immunostaining and biotinylation
S2R+ cells were transfected with Gyc76C constructs for 2 days. For live cell
surface immunostaining, transfected cells were blocked in ice-cold 10%
FBS/S2 medium on ice for 10 minutes, incubated with anti-Myc 9E10
(1:100)/10%FBS/S2 medium on ice for 30 minutes and washed with ice-
cold 3% sucrose/PBS. Cells were fixed in 4% paraformaldehyde/3% sucrose
for 10 minutes, permeabilized with 0.5% triton/PBS for 5 minutes, blocked
in 10% NGS/PBS for 15 minutes and incubated with rabbit anti-Myc 71D10
(1:500; Cell Signaling) overnight at 4°C. Secondary antibodies were
Alexa 488-conjugated goat anti-mouse IgG (1:500) and Alexa647-
conjugated goat anti-rabbit IgG (1:500; Molecular Probes). In biotinylation
cell surface protein assays, transfected cells were washed twice with ice-cold
PBS, incubated with 1 mg/ml EZ-link Sulfo-NHS-SS-Biotin (Thermo
Scientific) on ice for 20 minutes, washed twice with ice-cold PBS and then
incubated with 50 mM glycine on ice for 10 minutes prior to lysis in RIPA
buffer and sonication. Homogenates were centrifuged at 23,000 g for
20 minutes at 4°C, supernatants were incubated with NeutrAdvin beads
(Thermo Scientific) for 2 hours at 4°C and washed four times with Wash
buffer [150 mM NaCl, 50 mM Tris (pH 8.0), 1 mM MgCl and 1% NP40].
Precipitates were analyzed by western blot using anti-Myc 9E10 (1:2000).
Yeast two-hybrid screen
Yeast protocols used standard techniques (Golemis et al., 1994; Terman et
al., 2002). The Gyc76C intracellular domain (amino acids 1451-1525) was
PCR amplified and inserted into yeast expression vector pEG202 (bait
vector) to generate KC1 bait. KC1 was introduced into yeast strain EGY48,
containing the β-galactosidase-expressing plasmid pJK103. Western analysis
of transformed yeast using anti-LexA (Invitrogen) confirmed expression of
appropriately sized bait protein (unpublished data), and an activation assay
showed that the bait did not activate transcription (K.C. and A.L.K.,
unpublished). A 0-24 hour Drosophila embryonic cDNA library was cloned
into the yeast expression vector pJG4-5. Greater than 2×106clones were
screened and interactions assessed using a visual β-galactosidase assay and
a test of growth in the absence of leucine. Interacting yeast clones were
selected, and standard protocols were used to recover the library vector and
sequence clones on both strands. Over 30 interactors were identified and
subjected to secondary screen using a different bait, KC2, lacking the PDZ-
binding motif (amino acids 1434-1521).
We thank Drs Djiane and Mlodzik for generously providing dGIPC antibody and
dGIPC mutant flies, DGRC and Bloomington Stock Centers for fly stocks, and
Afshan Ismat for assistance with ISH experiments. We thank S. Jeong and X. Xie
for critical reading of the manuscript.
The authors declare no competing financial interests.
K.C. and A.L.K. designed experiments; K.C. performed experiments; K.C. and
A.L.K. analyzed data and wrote the manuscript.
This work was supported by The National Institutes of Health [NS35165] to A.L.K.
A.L.K. is an Investigator of the Howard Hughes Medical Institute. Deposited in
PMC for release after 6 months.
Supplementary material available online at
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