© 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 ARTICLEDevelopment (2014) doi:10.1242/dev.095968
exhibit a higher penetrance of motor axon defects than UAS:Sema-
1a/+ embryos (Fig. 1D and Table 1), indicating the presence of a
modest dominant phenotype resulting from heterozygosity at
Gyc76Cex173. The strong suppression of Sema-1a gain-of-function
motor axon phenotypes by removing one Gyc76C or PlexA allele
suggests Gyc76C participates in Sema-1a-PlexA-mediated repulsive
guidance in vivo.
Gyc76C physically associates with PlexA both in vitro and
We next performed co-immunoprecipitation (co-IP) experiments in
vitro using a Drosophila embryonic cell line, and in vivo using
transgenic fly lines. Myc-tagged full-length (FL) Gyc76C (Myc-FL
Gyc76C) and/or hemagglutinin (HA)-tagged FL PlexA (HA-FL
PlexA) were overexpressed in the adherent Drosophila S2R+ cell
line (Schneider, 1972). Immunoprecipitation of Myc-FL Gyc76C
using anti-Myc robustly co-immunoprecipitated HA-FL PlexA
(Fig. 2A, lane 1). Immunoprecipitation of HA-FL PlexA using anti-
HA precipitated Myc-FL Gyc76C (Fig. 2B, lane 3), and HA-FL
PlexA or Myc-FL Gyc76C was not immunoprecipitated by anti-Myc
or anti-HA, respectively (Fig. 2A, lane 3 and Fig. 2B, lane 2). We
did not detect interactions between HA-FL PlexA and Myc-tagged
dumbfounded (Duf), an immunoglobulin domain transmembrane
protein required for muscle fusion (Ruiz-Gómez et al., 2000), in
S2R+ cells (Fig. 2A, lane 5 and Fig. 2B, lane 5), demonstrating
specificity in our immunoprecipitation experiments. We generated
transgenic flies expressing HA-FL PlexA with, or without, Myc-FL
Gyc76C in all neurons using the UAS-GAL4 system (Brand and
Perrimon, 1993) and the neuronal driver elav-Gal4. Using anti-Myc
or anti-HA, we confirmed the expression of each transgene in
developing embryos (supplementary material Fig. S1). In embryonic
lysates generated from these transgenic flies, we observed robust co-
IP of Myc-FL Gyc76C using anti-HA (Fig. 2C, lane 2) only in flies
expressing both HA-FL PlexA and Myc-FL Gyc76C. Therefore,
Gyc76C and PlexA can form a protein complex both in vitro and in
To define region(s) of Gyc76C and PlexA that mediate association
between these proteins, we generated three truncated HA-PlexA
constructs: the PlexA extracellular region with transmembrane
domain (HA-PlexA EctoTM), the PlexA intracellular region with
transmembrane domain (HA-PlexA EndoTM) and the PlexA
intracellular region without transmembrane domain (HA-PlexA
Endo). Each truncated PlexA construct was expressed with, or
without, Myc-FL Gyc76C in S2R+ cells. Myc-FL Gyc76C
immunoprecipitated each of these HA-PlexA constructs (Fig. 2D,
lanes 5-8). Since PlexA constructs were expressed at different levels,
we normalized each interaction between Gyc76C and PlexA to the
corresponding PlexA expression level for that construct; this shows
that the extracellular region of PlexA alone interacts with Gyc76C
as strongly as FL PlexA, but suggests that the intracellular
associations, though present, may be weaker (Fig. 2D; K.C. and
A.L.K., unpublished). These experiments show that Gyc76C and
PlexA can physically associate and suggest this association involves
both extracellular and intracellular regions of these proteins.
Full-length Gyc76C exhibits guanylyl cyclase activity in
vitro, and this activity is influenced by each Gyc76C protein
We generated a series of N-terminally Myc-tagged Gyc76C deletion
constructs, each including one or two discrete protein domain
deletions (Fig. 3A). Each of these Gyc76C constructs was expressed
robustly in S2R+ cells (supplementary material Fig. S2A). Using a
direct cGMP enzyme immunoassay (EIA) (Materials and methods),
we measured total cGMP levels in S2R+ cells expressing wild-type
Myc-FL Gyc76C or Myc-Gyc76C[D945A], in which a crucial
amino acid (D945) in the guanylyl cyclase domain required for
catalytic activity and semaphorin-mediated axon guidance in
Drosophila (Thompson and Garbers, 1995; Ayoob et al., 2004) is
mutated. We observed significant cGMP levels in S2R+ cells
expressing Myc-FL Gyc76C but no detectable cGMP in cells
expressing Myc-Gyc76C[D945A] (Fig. 3B), confirming Drosophila
Gyc76C functions as a guanylyl cyclase.
Next, we assessed Gyc76C deletion constructs and found that
each Gyc76C protein domain influences Gyc76C-mediated cGMP
production in vitro (Fig. 3B). The Myc-Gyc76C construct that lacks
the entire cytoplasmic domain (ΔEndo) did not produce cGMP
levels above background (Fig. 3B). Deletion of the PDZ-binding
motif (PBM) (ΔPBM, deleting the four C-terminal amino acids)
completely abolished Gyc76C guanylyl cyclase (GC) activity.
Deletion of the entire extracellular domain (ΔEcto) resulted in
elevated cGMP production compared with FL Gyc76C, and this was
abolished when either the TM, or the PBM, was also deleted.
Deletion of the KHD (ΔKHD) resulted in greatly elevated GC
activity compared with FL Gyc76C, consistent with previous studies
Table 1. ISNb defect phenotypes in Sema-1a GOF in different genetic backgrounds
Total defects (n)*
Distinct ISNb defects
*n=total number of hemisegements scored. Abnormal ISNb stalling phenotypes are defined as a failure of ISNb axons to innervate ventral lateral muscles
(between 12/13 or 6/7). Phenotypes include weak, or absent, innervation between muscles 12/13 and between muscles 6/7, muscle target bypasses and
axon positioning defects.
‡Bypass phenotypes, including parallel bypass and fusion bypass events, are defined as failure of ISNb axons to enter the ventral muscle field and extend
dorsally in close proximity to the ISN or along the ISN, resulting in a thicker ISN bundle.
§Positioning defects are defined as ISNb axon bundles growing dorsally along the ISN but with RP3 or RP5 still innervating ventral muscles.
¶Significantly different from values for UAS:Sema-1a/+, Mef2-Gal4/+ embryos. Z-test for two proportions; P<0.005.
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
demonstrating that the mammalian KHD negatively regulates cGMP
production (Chinkers and Garbers, 1989; Potter, 2011). cGMP levels
produced by Gyc76C lacking both KHD and PBM domains
(ΔKHD+PBM) were greatly reduced compared with ΔKHD alone;
however, these cGMP levels were significantly higher than FL
Gyc76C. Deletion of the dimerization domain (ΔDD) abolished all
GC, like vertebrate receptor GCs (Chinkers and Garbers, 1989).
Finally, deletion of the unique Gyc76C C-terminal domain (ΔCterm)
yielded very low cGMP levels, and deletion of both Cterm and PBM
abolished GC activity (Fig. 3B).
Protein expression levels and localization could contribute to
differences in GC activity. We assessed total protein levels for all
Gyc76C constructs using western blot analyses (supplementary
material Fig. S2A), and also determined cell surface protein
expression levels using live cell surface immunostaining
(supplementary material Fig. S2B). Some of these modified Gyc76C
proteins exhibited different total protein expression levels, and also
some showed more robust cell surface localization than others. For
example, both ΔEcto and ΔKHD proteins are robustly localized to the
cell surface (supplementary material Fig. S2B, top) and exhibit high
GC activity (~5-10 times FL Gyc76C). However, total protein
expression levels produced by these constructs are comparable with,
or lower than, FL Gyc76C (supplementary material Fig. S2A, lanes 2,
4 and 7). Several constructs that show low-to-no GC activity do show
robust cell surface localization (Gyc76C[D945A], ΔCterm,
ΔCterm+PBM and ΔEndo; supplementary material Fig. S2B).
Deletion of the PBM alone, or with the Ecto or KHD domain, does
not significantly alter total protein expression levels compared with
FL Gyc76C (supplementary material Fig. S2A, lanes 2, 5, 8, and 13);
however, it does reduce cell surface protein localization
(supplementary material Fig. S2B,
ΔKHD+PBM shows robust GC activity (~3 times FL Gyc76C). We
observed low cell surface protein localization and total protein for the
ΔDD construct (supplementary material Fig. S2B, middle). For
ΔEcto+TM, total protein levels are high; however, as expected, this
variant shows no cell surface localization (supplementary material
Fig. S2B, bottom). Biochemical assessments of cell-surface protein
levels employing biotinylation of cell-surface proteins revealed
protein levels for each Gyc76C deletion variant commensurate with
cell-surface labeling experiments (K.C. and A.L.K., unpublished).
Therefore, though there are differences in total protein expression and
cell-surface localization of Gyc76C variants, except for ΔDD, these
do not account for the differences in GC activity we observe among
these Gyc76C proteins. ΔCterm and ΔCterm+PBM show robust total
Fig. 2. Gyc76C physically associates
with PlexA, both in vitro and in vivo.
(A,B) Lysates from Drosophila S2R+
cells expressing full-length (FL) Myc-
Gyc76C with, or without, FL HA-PlexA
were immunoprecipitated using anti-Myc
and blotted with anti-HA to detect the
presence of HA-PlexA (A, lane 1). IPs
were also performed using anti-HA and
blotted with anti-Myc to detect the
presence of Myc-Gyc76C (B). FL Myc-
PlexB immunoprecipitates FL HA-PlexA
(lane 4, positive control), whereas Myc-
Duf fails to immunoprecipitate FL HA-
PlexA (lane 5, negative control). FL HA-
PlexA immunoprecipitates FL Myc-
Gyc76C (lane 3); however, FL PlexA fails
to immunoprecipitate Myc-Duf (lane 5,
negative control). 0.5% (3.75 μl/lane) of
total cell lysate (750 μl) was loaded for
the input; 20% (10 μl/lane) of the total
immunoprecipitate (50 μl) was loaded for
the IP. (C) Lysates from embryos
expressing Myc-Gyc76C under the
control of elav-Gal4 with, or without, HA-
PlexA were immunoprecipitated using
anti-Myc and blotted with anti-HA to
detect the presence of HA-PlexA (lane
2). 0.5% (2.5 μl/lane) of total embryo
lysate (500 μl) was loaded for the input;
20% (10 μl/lane) of the total
immunoprecipitate (50 μl) was loaded for
the IP. (D) Lysates from S2R+ cells
expressing FL Myc-Gyc76C with, or
without, FL HA-PlexA (or several PlexA
deletion constructs) were
immunoprecipitated using anti-Myc and
then blotted with anti-HA to detect the
presence of HA-PlexA constructs. 0.5%
(3.75 μl/lane) of the total embryonic
lysate (750 μl) was loaded for the input;
25% (10.25 μl/lane) of the total
immunoprecipitate (50 μl) was loaded for
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
and cell-surface protein expression but produce no cGMP, suggesting
that specific molecular mechanisms regulate Gyc76C cyclase activity.
These experiments show that each Gyc76C protein domain influences
cGMP production and suggest Gyc76C GC activity is regulated by
both extracellular and intracellular mechanisms.
Gyc76C protein domain requirements for motor axon
guidance rescue in Gyc76C mutant embryos
To investigate Gyc76C protein domain function in vivo, we generated
12 different Gyc76C transgenic fly lines (two independent fly lines
per transgene), each expressing an altered Gyc76C lacking a different
protein domain (ΔEcto, ΔKHD, ΔDD, ΔCterm, ΔEndo and ΔPBM).
Each transgene included an N-terminal Myc, and we drove neuronal
expression of these transgenes to determine the ability of each to
rescue Gyc76C motor axon defects (Fig. 4A). Gyc76C protein
expression levels in each transgenic line, assessed using quantitative
western blot analysis, revealed that these constructs vary somewhat in
their expression levels. However, each pair of independent transgenic
lines harboring the same Gyc76C deletion construct showed
comparable expression, and in all cases total protein levels were either
equal to, or significantly greater than, those observed for FL Gyc76C
(supplementary material Fig. S3A). Therefore, variations in expression
of the deletion constructs are not due to positional effects on transgene
expression. Similar to ISNb motor axon pathways, SNa axons also
display stereotypic neuromuscular connectivity; they navigate past the
ventral longitudinal muscle field to innervate lateral transverse
muscles 22, 23 and 24 (Landgraf et al., 1997), and together with the
ISNb allow for assessment of motor axon guidance in vivo (Araújo
and Tear, 2003). Gyc76C mutant embryos show significant defects in
these motor axon pathways (Ayoob et al., 2004). These defects are not
a secondary consequence of longitudinal muscle defects because
overall muscle organization in Gyc76C mutant embryos is apparently
normal (supplementary material Fig. S3B,B′). Furthermore, Gyc76C
expression in somatic muscles fails to rescue motor axon guidance
defects in Gyc76C homozygous mutants (K.C. and A.L.K.,
The only Gyc76C transgenic flies that rescue the ISNb and SNa
defects observed in Gyc76C-null mutants are the two independent
FL Gyc76C transgenes [FL(65) and FL(5.2)]. In these flies, ISNb
and SNa pathway defects observed in Gyc76Cex173-null mutants
were rescued from 42% to 21%, and from 39% to 20%, respectively
(Fig. 4A, red highlight; P<0.005; comparable with previous
experiments; Ayoob et al., 2004). All other Gyc76C deletion
constructs failed to rescue ISNb or SNa defects (Fig. 4A). ΔDD,
ΔCterm, ΔEndo and ΔPBM exhibit no GC activity in in vitro GC
assays, and so elevated expression levels of proteins encoded by
these constructs, compared with FL Gyc76C (supplementary
material Fig. S3B, lanes 6-13), are not likely to result in elevated
Fig. 3. Full-length Gyc76C exhibits guanylyl
cyclase activity, and this activity is influenced
by each Gyc76C protein domain. (A) Protein
domain organization of FL Gyc76C and deletion
constructs used in this study. D945 is the
conserved aspartate residue required for guanylyl
cyclase catalytic activity. (B) Normalized total
cGMP levels in S2R+ cells expressing FL Gyc76C
or Gyc76C deletion constructs (transfections
performed in triplicate, cells were harvested 2 days
after transfection). 5×Myc was tagged to the N
terminus of each construct. cGMP levels were first
measured in lysates derived from cells expressing
each transgene and then normalized to the protein
expression level of FL Gy76C, as determined by
western blot analysis (Materials and methods).
RESEARCH ARTICLE Development (2014) doi:10.1242/dev.095968
cGMP production. By contrast, ΔEcto and ΔKHD exhibit the
highest in vitro GC activities, yet these two constructs do not rescue
the motor axon defects in Gyc76C mutants; ISNb and SNa defects
are phenotypically comparable, both quantitatively and qualitatively,
with those observed in Gyc76C-null mutants (Ayoob et al., 2004),
suggesting that motor axon pathfinding defects in unrescued
embryos are not the result of dominant-negative effects from
Taken together, these results suggest that Gyc76C cyclase activity
is regulated by both extracellular and intracellular protein domains.
The unique Gyc76C C terminus, and also the PBM, are crucial for
normal cyclase activity in vitro, suggesting that signaling components
exist that interact with Gyc76C and regulate its function.
PlexA augments cGMP levels produced by Gyc76C in vitro
We next investigated whether the PlexA receptor influences Gyc76C
GC activity, employing the direct cGMP EIA assay described above
and a different Drosophila cell line, DmBG2, to assess whether or
not a functional relationship exists between Gyc76C and PlexA.
Unlike S2R+ cells, which are derived from a macrophage-like
lineage, DmBG2 cells are derived from the Drosophila third instar
larval CNS (Yanagawa et al., 1998). A constant amount of the
construct encoding FL Gyc76C DNA (or CD8-GFP as a control),
0.04 μg, was co-transfected into DmB2 cells with 0.2 μg (5×), 0.04
μg (1×) or 0.008 μg (1/5×) of the construct encoding FL PlexA
DNA. The amount of PlexA DNA transfected into DmBG2 cells in
these experiments directly correlates with PlexA protein levels
(supplementary material Fig. S4, top panel). When fivefold more
PlexA DNA compared with Gyc76C DNA was transfected into
DmBG2 cells, total cGMP levels were increased by ~2.4-fold over
what we observed for Gyc76C alone (Fig. 5A, representative
experiment; Fig. 5B, average fold change, four independent
experiments). In the absence of transfected FL Gyc76C, FL PlexA
alone had no significant effect on cGMP levels in DmBG2 cells.
Different amounts of PlexA DNA transfected into DmBG2 cells in
these experiments did not affect Gyc76C protein expression levels
(supplementary material Fig. S4, bottom panel, lanes 1-4). We
performed similar GC assays with ΔEcto, ΔKHD or ΔPBM;
however, 5×PlexA did not increase GC activity of these constructs
(K.C. and A.L.K., unpublished). PlexA-mediated augmentation of
Gyc76C GC activity is consistent with our genetic analyses showing
that Gyc76C influences Sema-1a gain-of-function phenotypes in the
PNS (this study), and also PlexA-dependent gain-of-function
phenotypes in the CNS (Ayoob et al., 2004), supporting a model
whereby Gyc76C facilitates Sema-1a/PlexA-mediated axon
A PDZ domain-containing protein, dGIPC, interacts with
Gyc76C and increases cell surface expression of Gyc76C
Our in vitro and in vivo results suggest that there may be PDZ
(PSD95/Dlg1/ZO-1) domain-containing proteins that interact with
Fig. 4. Gyc76C protein domain requirements for
rescue of motor axon guidance defects in the
Gyc76C loss-of-function mutants.
(A) Quantification of ISNb and SNa phenotypes in the
Gyc76C loss-of-function mutant embryos and in
rescue experiments using various Gyc76C deletion
constructs. †ISNb phenotypes: failure of RP5 or RP3
ISNb axons to innervate ventral longitudinal muscles
12/13 or 6/7. ‡SNa phenotypes: failure of SNa axons
to defasciculate at choice points, to reach muscle 24
and/or to project along appropriate trajectories. N,
number of hemisegments scored per genotype.
*Significantly different from Gyc76Cex173homozygous
mutants. Z-test for two proportions; P<0.005. (B) The
relationship between in vitro GC activity and in vivo
rescue ability of FL Gyc76C and various domain
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
Gyc76C and regulate its function. We used the C-terminal 75 amino
acids of Gyc76C, which include the PDZ-binding motif, as a bait
(KC1) to search for Gyc76C-interacting proteins, screening a
Drosophila embryonic (0-24 hours) yeast two-hybrid (Y2H) cDNA
library (supplementary material Fig. S5A; Materials and methods).
Clones encoding interacting proteins were further examined using
another bait (KC2) that is similar to KC1 but lacks the PDZ-binding
motif (supplementary material Fig. S5A). We found that clones that
interacted with KC1 but not KC2 encoded the Drosophila GAIP
interacting protein, C terminus homolog (dGIPC; Kermit – FlyBase)
(Djiane and Mlodzik, 2010).
There are three mammalian, two Xenopus and one Drosophila
GIPC. GIPC is an intracellular protein with a centrally located PDZ
domain and no other conserved sequence motifs. GIPC interacts
with RGS-GAIP (regulator of G protein signaling-GTPase activating
protein for Gαi) (De Vries et al., 1998) and also with other GIPC-
binding partners (Cai and Reed, 1999; Wang et al., 1999; Lou et al.,
2001; Tan et al., 2001). GIPC is implicated in regulating the
distribution of the Sema-5A protein (Wang et al., 1999) and NMDA
receptor trafficking (Yi et al., 2007) in vitro. The Drosophila GIPC
homolog, dGIPC, was first described in a gain-of-function screen
designed to identify planar cell polarity genes in Drosophila (Djiane
and Mlodzik, 2010). dGIPC is also important for locomotor activity
and longevity, possibly through the regulation of dopamine (DA)
receptor trafficking (Kim et al., 2010).
We performed in vitro co-IP experiments, expressing HA-dGIPC
with Myc-FL Gyc76C, Myc-ΔPBM, Myc-ΔCterm, Myc-
Immunoprecipitation of HA-dGIPC with anti-HA revealed a robust
interaction between Gyc76C and dGIPC. This was greatly
attenuated when the Gyc76C PBM domain was removed (Fig. 6A,
lanes 1 and 2, asterisk), and was completely abolished when the
Gyc76C C-terminal region, the Gyc76C C-terminal region plus the
PBM or the entire intracellular region of Gyc76C was deleted
(Fig. 6A, lanes 3-5). These experiments demonstrate that dGIPC
interacts with Gyc76C in vitro, requiring the PBM domain and, to a
much lesser extent, other Gyc76C C-terminal sequences.
Since mammalian GIPC regulates the distribution of several
transmembrane proteins (Wang et al., 1999; Tan et al., 2001; Yi et
al., 2007), we asked whether dGIPC influences cell surface
localization of Gyc76C. Using biotinylation cell surface protein
labeling assays, we found a significant increase in the levels of
Gyc76C protein localized to the plasma membrane of S2R+ cells
overexpressing dGIPC (Fig. 6B, lanes 2 and 4). This result suggests
that dGIPC regulates Gyc76C GC activity by influencing Gyc76C
cell surface distribution.
or GFP in S2R+ cells.
Fig. 5. PlexA augments Gyc76C cGMP
production in vitro. (A) Quantification of a
single experiment showing normalized cGMP
production resulting from transfection with FL
Gyc76C and varying levels of FL PlexA in
DmBG2 cells grown for 3.5 days in culture.
0.2 μg (5×), 0.04 μg (1×) or 0.008 μg (1/5×)
of FL PlexA cDNA was transfected with a
constant amount (0.04 μg) of FL Gyc76C (or
CD8-GFP as a control). Average cGMP
levels were determined from cell lysates
(transfections performed in triplicate) and
normalized to Myc-FL Gyc76C expression
(see Materials and methods). Error bars
indicate s.d. of cGMP levels in triplicate
samples from this representative GC assay.
*Significantly different from cGMP levels
measured in 1×FL Gyc76C alone (t-test for
two proportions; P<0.05). (B) Fold change in
normalized cGMP production determined
from four independent GC assays, as
described in A. Error bars indicate s.d. for
each condition for cGMP production
determined from four independent GC
assays. **Significantly different from 1×FL
Gyc76C alone (t-test for two proportions;
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
dGIPC is required for embryonic motor axon guidance and
genetically interacts with Gyc76C, Sema1a and PlexA
We next examined dGIPC function in motor axon guidance and
observed significant defects in the ISNb (46.1%) and SNa (31.5%)
motor axon pathways in embryos trans-heterozygous for two
chromosomal deficiency lines (Df7890 and Df8941) that remove
nine genes, including dGIPC (K.C. and A.L.K., unpublished). The
dGIPCex31allele does not express dGIPC protein (Djiane and
Mlodzik, 2010), and dGIPCex31homozygous embryos show
pathfinding defects in ISNb (35.5%) and SNa (30.4%) pathways;
dGIPCex31/Df8941 embryos exhibit 46.2% and 20.8% pathfinding
defects in ISNb and SNa pathways, respectively. These phenotypes
are qualitatively and quantitatively similar to those observed in
dGIPCex31homozygous embryos. Furthermore, the predominant
pathfinding defects are qualitatively similar to those we observed in
Gyc76C homozygous mutant embryos, though the penetrance of
SNa defects is somewhat lower. Together, these results show that the
dGIPCex31allele is likely a null, or strong hypomorphic, allele of
Embryos heterozygous for dGIPC and either Gyc76C, Sema-1a
or PlexA, were assessed for trans-heterozygous genetic interactions
to determine whether or not these genes function in the same genetic
pathway (Artavanis-Tsakonas et al., 1995; Winberg et al., 1998b).
dGIPCex31/+; Gyc76Cex173/+ embryos show 48.5% and 23.1%
defects in ISNb and SNa pathways, respectively (Fig. 7A). dGIPC
also genetically interacts with Sema-1a and PlexA, as observed in
dGIPCex31/+, Sema-1aP1/+ and dGIPC ex31/+;; PlexADf(4)3C/+
embryos (Fig. 7A). In these embryos, ISNb axons often fail to
innervate their muscle targets with a penetrance comparable to that
observed in dGIPCex31homozygotes.
Qualitatively and quantitatively these trans-heterozygous mutant
embryos display ISNb phenotypes similar to dGIPC or Gyc76C
homozygous mutant embryos, including stalling defects at M12/13
and at M6/7 (Fig. 7A). However, in these embryos we observe only
mild SNa defects (Fig. 7A), suggesting that alterations in signaling
in these trans-heterozygous embryos are not strong enough to affect
all motor axon pathways. We also analyzed motor axon pathways in
dGIPCex31; Gy76Cex173double null mutants and observed that the
penetrance of ISNb defects is similar to that observed in dGIPCex31
or Gyc76Cex173homozygous single mutants, whereas the penetrance
of SNa defects is similar to dGIPCex31homozygous mutants
(Fig. 7A, red box). These data suggest that dGIPC and Gyc76C
function in the same genetic pathway, and together our genetic
analyses support dGIPC functioning with Gyc76C in Sema-
Neuronal dGIPC is required for embryonic motor axon
dGIPC exhibits strong expression in CNS midline glia of the
Drosophila ventral nerve cord (VNC) (Djiane and Mlodzik, 2010).
In situ hybridization (ISH) with a dGIPC-specific cRNA antisense
probe, and immunohistochemistry (IHC) using an antibody
directed against dGIPC, revealed enriched dGIPC in the midline
region of VNC wild-type embryos. This midline staining was
absent when a dGIPC sense probe was used in ISH (K.C. and
A.L.K., unpublished), or when dGIPCex31-null embryos were used
in IHC experiments (Djiane and Mlodzik, 2010). However, we
also detected weak, but significant, dGIPC protein expression in
ventral nerve roots exiting the VNC (supplementary material
Fig. S5B, red arrowheads) that is not present in dGIPC-null
mutants (supplementary material Fig. S5B′). dGIPC-null mutants
display motor axon defects, and so dGIPC could function in
neurons, non-neuronal cells, or both. In the Drosophila brain,
dGIPC is predominantly expressed in glial cells but is also
expressed in DA neurons, and DA neuronal expression of dGIPC
is crucial for locomotor activity (Kim et al., 2010).
To determine dGIPC cell type requirements, we employed Gal4
drivers for in vivo rescue experiments dGIPCex31homozygous
mutant embryos. We expressed dGIPC in all neurons using elav-
Gal4 in dGIPCex31-null mutant embryos and found significant
rescue of both ISNb and SNa motor axon guidance defects
(Fig. 7B,B′): from 35.5% to 17.7% and from 30.4% to 15.1%,
Fig. 6. dGIPC physically interacts with Gyc76C and
increases Gyc76C cell surface localization in vitro.
(A) CoIP of S2R+ cell lysates expressing HA-dGIPC
with, or without, FL Myc-Gyc76C. (B) Biotinylation of
S2R+ cells expressing FL Myc-Gyc76C and HA-dGIPC
prior to cell lysis. Two independent experiments (I, II)
show a significant increase of biotinylated Gyc76C with
dGIPC (compare lanes 1 and 3 with lanes 2 and 4).
RESEARCH ARTICLE Development (2014) doi:10.1242/dev.095968
respectively (Fig. 7B). When dGIPC was expressed in all glial
cells using the repo-Gal4 (Sepp et al., 2001) driver, or in midline
glial cells and MP1 CNS neurons using the single-minded GAL4
(sim-GAL4) driver (Hidalgo and Brand, 1997), no rescue of ISNb
defects (36.3% for repo-Gal4; 31.9% for sim-Gal4), and seemingly
apparent but not statistically significant rescue of SNa defects
(21.8% for repo-Gal4; 20.5% for sim-Gal4), were observed in
dGIPCex31-null mutant embryos carrying repo-Gal4 or sim-Gal4
(Fig. 7B,B″). As elav-Gal4 also drives expression in neural
progenitors and embryonic glia (Berger et al., 2007), we employed
two additional neuron-specific drivers: scabrous-GAL4 (sca-Gal4)
(Mlodzik et al., 1990) and OK371-Gal4 (Mahr and Aberle, 2006).
Neuronal expression of dGIPC driven by sca-Gal4 also rescues
both ISNb (13.4%) and SNa (15.3%) motor axon defects in
dGIPCex31homozygous mutant embryos (Fig. 7B). In OK371-
Gal4/+, dGIPCex31; UAS:dGIPC/+
significant rescue of the ISNb pathway (19.5%; P<0.005), and
seemingly apparent but not statistically significant rescue of the
SNa pathway (20.9%; P>0.005). Therefore, it remains uncertain
whether dGIPC expression in glia or neurons rescues SNa defects
in dGIPC mutants. However, neuronal dGIPC expression is
required for proper ISNb pathfinding. These results show that
dGIPC robustly associates with the PBM domain of Gyc76C, that
embryos, we observe
it is required for embryonic motor axon pathfinding, and that
dGIPC strongly interacts with Gyc7C, Sema-1a and PlexA.
We provide here support for the Drosophila receptor guanylyl
cyclase Gyc76C being a component of the Sema1a-PlexA signaling
cascade in vivo. Each discrete Gyc76C protein domain is essential
for Gyc76C catalytic activity in vitro and for motor axon guidance
in vivo. Furthermore, the cytosolic protein dGIPC interacts with
Gyc76C and functions in Gyc76C-mediated motor axon guidance.
These results provide an in vivo link between semaphorin-mediated
repulsive guidance and alteration of intracellular cGMP levels.
Gyc76C is part of the Sema-1a-PlexA signaling complex
The Gyc76C-Sema-1a gain-of-function genetic interactions we
observe here are consistent with previous observations showing that
Gyc76C loss and gain of function modifies aberrant CNS midline
crossing by FasII+longitudinal axons in a PlexA gain-of-function
genetic background (Ayoob et al., 2004). Furthermore, we observe
robust physical interactions between Gyc76C and PlexA both in
vitro and in vivo, raising the possibility that PlexA regulates
Gyc76C-mediated signaling. Co-expressing PlexA at high levels in
vitro augments cGMP levels produced by Gyc76C. Future work will
Fig. 7. dGIPC genetically interacts
with Gyc76C, Sema-1a and PlexA, and
is required in neurons for motor axon
pathfinding. (A) Quantification of motor
axon defects in dGIPC homozygous
mutant embryos and genetic interactions
between dGIPC and Gyc76C, Sema-1a
or PlexA. Motor axon defects in dGIPC;
Gyc76C homozygous double mutants
are highlighted (red box). Numbers in
parentheses, total number of
hemisegments scored for ISNb or SNa
pathways. (A′) Filleted late stage 16
embryo stained with1D4 revealing motor
axons in abdominal segments. In a
dGIPCex31homozygous mutant embryo,
axons within the ISNb (black arrows) and
SNa (black arrowheads) pathways often
fail to reach their proper targets. Scale
bar: 5 μm. (B) Quantification of rescue
experiments using dGIPC driven by cell
type-specific Gal4 drivers. **Significantly
different from dGIPCex31homozygous
mutants. Z-test for two proportions;
P<0.005. N.S., not significantly different
from values for dGIPCex31homozygous
mutants. (B′,B″) Filleted late stage 16
embryos stained with 1D4 revealing
motor axons in abdominal segments.
(B′) Neuronal dGIPC in a dGIPCex31
mutant restores proper ISNb (black
arrows) and SNa (black arrowhead)
muscle innervation. (B″) Glial expression
of dGIPC does not rescue ISNb (open
arrows) or SNa muscle innervation (open
arrowhead). Scale bar: 5 μm. Anterior is
leftwards; dorsal is upwards.
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
establish whether extracellular, intracellular, or both, types of
protein-protein associations between Gyc76C and PlexA are
essential for regulating Gyc76C enzymatic activity.
Each Gyc76C domain is required for cyclase activity and
proper motor axon guidance
Our Gyc76C structure-function analyses are consistent with the idea
that PlexA binds to the extracellular and intracellular regions of
Gyc76C to relieve inhibitory effects on GC activity from of Gyc76C
intramolecular interactions, increasing cGMP levels within
extending motor axon growth cones and affecting growth cone
guidance. This is reminiscent of Sema-3A bath application
increasing intracellular cGMP levels in Xenopus spinal neurons in
vitro (Togashi et al., 2008), and our results suggest that intracellular
cGMP produced by Gyc76C is required for Sema-1a-mediated
repulsion. However, it is possible that signaling by intracellular
cGMP is coupled with intracellular cAMP in Sema-1a-mediated
repulsive guidance events (Nishiyama et al., 2003), and future work
will determine whether varying the cAMP-to-cGMP ratio modulates
Sema-1a-mediated repulsion, or converts it to attraction. Bath
application of Sema-1a did not affect Gyc76C-PlexA physical
associations or Gyc76C-PlexA-mediated cGMP production (K.C.
and A.L.K., unpublished), suggesting that Sema-1a-dependent
regulation of intracellular cGMP levels could involve ligand-gated,
dynamic, spatiotemporal regulation of GC activity; visualizing this
signaling event will require real-time imaging of cGMP during
repulsive growth cone steering.
The small GTPase Rac and its downstream effector p21 activated
kinase (PAK) can regulate receptor GCs to raise cellular cGMP
levels in fibroblasts in vitro, and the kinase domain of PAK interacts
with the cyclase domain of receptor GC-E (Guo et al., 2007; Guo et
al., 2010); PAK, therefore, may associate with Gyc76C and regulate
this receptor GC during axon pathfinding.
dGIPC interacts with Gyc76C and is required for motor axon
guidance in Drosophila
The deletion of the Gyc76C PBM strongly suppresses cGMP
production by FL Gyc76C, and Gyc76C variants lacking the PBM
motif exhibit low cell-surface expression levels, suggesting dGIPC
regulates Gyc76C cell-surface localization. PDZ-containing proteins
could form a protein scaffold required for plasma membrane
localization of the Sema-1a-PlexA/Gyc76C signaling complex, and
we find that the PDZ domain-containing dGIPC protein interacts
with Gyc76C. In vertebrates, GIPC regulates protein trafficking,
subcellular localization and various signaling events (De Vries et al.,
1998; Cai and Reed, 1999; Wang et al., 1999; Lou et al., 2001; Tan
et al., 2001; Yi et al., 2007). Gyc76C cell-surface localization is
enhanced in vitro in the presence of dGIPC, and this may serve to
regulate Gyc76C-mediated signaling. dGIPC genetic analyses show
that dGIPC plays a neuronal role in motor axon guidance, and this
likely occurs through interactions that modulate Gyc76C-mediated
cGMP signaling. Mammalian GIPC forms dimers and multimeric
complexes (Bunn et al., 1999; Gao et al., 2000; Hirakawa et al.,
2003; Jeanneteau et al., 2004; Kedlaya et al., 2006; Naccache et al.,
2006; Varsano et al., 2006). dGIPC may be a part of a molecular
scaffold that couples Gyc76C to cell membrane trafficking
machinery or anchors Gyc76C to the plasma membrane.
Alternatively, dGIPC may be essential for activating or transducing
Gyc76C-mediated cGMP signaling in axon guidance events. Future
genetic and biochemical analyses will reveal the downstream
signaling components that respond to changes in cGMP levels in
vivo and direct discrete neuronal growth cone steering responses.
MATERIALS AND METHODS
Drosophila strains and phenotypic characterization
Culturing Drosophila was performed as described previously (Yu et al.,
1998; Terman et al., 2002; Ayoob et al., 2004). Stocks Df7860 and Df8941
were from the Bloomington Stock Center. Gal4 drivers were: elav(2)-GAL4,
elav(3E)-GAL4 (Yao and White, 1994), Mef2-Gal4 (Ranganayakulu et al.,
1996), sim-Gal4 (Hülsmeier et al., 2007), repo-Gal4 (Sepp et al., 2001),
OK371-Gal4 (Ramadan et al., 2007) and Sca-GAL4 (Klaes et al., 1994).
Analyses of axon guidance defects performed as described previously (Yu
et al., 1998).
Gyc76C deletion constructs
Gyc76C (amino acids 1-1525) domains are defined as Ecto (1-492), TM
(494-514), KHD (517-812), DD (815-870), Cterm (1105-1521), Endo (517-
1525) and PBM (1522-1525). PlexA (amino acids 1-1945) domains are
defined as: EctoTM(1-1330), TM (1282-1300), EndoTM(1271-1945) and
Endo (1305-1945). Endogenous Gyc76C or PlexA signal peptide replaced
with Igk-leader sequence, followed by 5×Myc (EQLISEEDL) or 2×HA
(YPYDVPDYA), respectively. dGIPC from a cDNA clone was C-terminally
tagged with 2×HA. Constructs were inserted into pUAST (Brand and
Perrimon, 1993); UAS-CD8-EGFP (pUAST-DEST16) obtained from the
Drosophila Genomics Resource Center.
S2R+ cells were grown in Schneider’s medium (Invitrogen) with 10% heat-
inactivated fetal bovine serum (FBS); ML-DmBG2 cells were grown in
Shields and Sang M3 medium with 10% heat-inactivated FBS and 10 μg/ml
insulin. Both cells types were cultured at 25°C [procedures can be found at
Drosophila S2R+ cells were transfected with pUAST:5×Myc- FL Gyc76C
with, or without, full-length pUAST:2×HA-FL PlexA using Effectene
(Qiagen). Forty-eight hours later, cell lysates were immunoprecipitated with
anti-Myc monoclonal antibody (9E10; Sigma) and blotted with anti-HA
monoclonal antibody at 1:2500 (12CA5; Roche); alternatively, cell lysates
were immunoprecipitated using anti- HA (12CA5) and immunoblotted using
anti-Myc (9E10) at 1:2500. For in vivo co-immunoprecipitation, UAS:HA-
PlexA, elav-Gal4/Cyo flies were crossed to either UAS:Myc-FL Gyc76C or
wild-type flies. Embryonic lysates were isolated and immunoprecipitated
with anti-Myc (9E10) (Terman et al., 2002). Immunoprecipitates were
probed using anti-HA (12CA5) at 1:2500 and anti-Myc (9E10) at 1:2500.
Guanylyl cyclase activity assay
In vitro cGMP concentrations determined from cell lysates with the Direct
cGMP Enzyme Immunoassay Kit (Assay Designs, # 900-014). S2R+ cells
and ML-DmBG2 cells were transfected for 36 hours or 72 hours,
respectively. Each well of a 24-well plate was plated with 0.5×106cells.
Transfected cells were incubated with 0.5 mM IBMX in serum-free medium
for 30 minutes at room temperature with gentle rocking, washed once with
1×PBS, lysed in 0.1 M HCl for 15 minutes at room temperature, and spun
at 600 g for 5 minutes. Supernatants were diluted four- or fivefold in 0.1 M
HCl for cGMP concentration determination. Expression of FL Gyc76C and
the Gyc76C construct variants in western blots were quantified using ImageJ
and normalized using actin controls. Raw cGMP levels produced by each
Gyc76C variant were then normalized to the protein expression level of FL-
Gy76C (raw cGMP levels × [(FL-Gy76C protein expression/corresponding
actin loading control)/(individual construct protein expression/
corresponding actin loading control)]).
Embryo collections and staining were performed as described (Yu et al.,
1998; Ayoob et al., 2004). Primary antibodies used were: anti-FasII mAb
1D4 (1:4; Van Vactor et al., 1993), rabbit anti-Sema-1a (1:500; Yu et al.,
1998), mouse anti-MHC (1:1000; Sigma), mouse anti-dGIPC (1:250; Djiane
and Mlodzik, 2010), anti-Myc 9E10 (1:500; Sigma) and anti-HA 12CA5
(1:500; Roche). HRP-conjugated goat anti-mouse and anti-mouse IgG/M
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
Araújo, S. J. and Tear, G. (2003). Axon guidance mechanisms and molecules:
lessons from invertebrates. Nat. Rev. Neurosci. 4, 910-922.
Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch signaling.
Science 268, 225-232.
Ayoob, J. C., Yu, H. H., Terman, J. R. and Kolodkin, A. L. (2004). The Drosophila
receptor guanylyl cyclase Gyc76C is required for semaphorin-1a-plexin A-mediated
axonal repulsion. J. Neurosci. 24, 6639-6649.
Bashaw, G. J. and Klein, R. (2010). Signaling from axon guidance receptors. Cold
Spring Harb. Perspect. Biol. 2, a001941.
Berger, C., Renner, S., Lüer, K. and Technau, G. M. (2007). The commonly used
marker ELAV is transiently expressed in neuroblasts and glial cells in the Drosophila
embryonic CNS. Dev. Dyn. 236, 3562-3568.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of
altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Bunn, R. C., Jensen, M. A. and Reed, B. C. (1999). Protein interactions with the
glucose transporter binding protein GLUT1CBP that provide a link between GLUT1
and the cytoskeleton. Mol. Biol. Cell 10, 819-832.
Cai, H. and Reed, R. R. (1999). Cloning and characterization of neuropilin-1-
interacting protein: a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with
the cytoplasmic domain of neuropilin-1. Neuroscience. 19, 6519-6527.
Chinkers, M. and Garbers, D. L. (1989). The protein kinase domain of the ANP
receptor is required for signaling. Science 245, 1392-1394.
Cho, J. Y., Chak, K., Andreone, B. J., Wooley, J. R. and Kolodkin, A. L. (2012). The
extracellular matrix proteoglycan perlecan facilitates transmembrane semaphorin-
mediated repulsive guidance. Genes Dev. 26, 2222-2235.
Davies, S. A. (2006). Signalling via cGMP: lessons from Drosophila. Cell. Signal. 18,
De Vries, L., Lou, X., Zhao, G., Zheng, B. and Farquhar, M. G. (1998). GIPC, a PDZ
domain containing protein, interacts specifically with the C terminus of RGS-GAIP.
Proc. Natl. Acad. Sci. USA 95, 12340-12345.
Desai, C. J., Gindhart, J. G., Jr, Goldstein, L. S. and Zinn, K. (1996). Receptor
tyrosine phosphatases are required for motor axon guidance in the Drosophila
embryo. Cell 84, 599-609.
Dickson, B. J. (2002). Molecular mechanisms of axon guidance. Science 298, 1959-
Djiane, A. and Mlodzik, M. (2010). The Drosophila GIPC homologue can modulate
myosin based processes and planar cell polarity but is not essential for
development. PLoS ONE 5, e11228.
Dontchev, V. D. and Letourneau, P. C. (2002). Nerve growth factor and semaphorin
3A signaling pathways interact in regulating sensory neuronal growth cone motility.
Neuroscience. 22, 6659-6669.
Gao, Y., Li, M., Chen, W. and Simons, M. (2000). Synectin, syndecan-4 cytoplasmic
domain binding PDZ protein, inhibits cell migration. J. Cell. Physiol. 184, 373-379.
Gibbs, S. M., Becker, A., Hardy, R. W. and Truman, J. W. (2001). Soluble guanylate
cyclase is required during development for visual system function in Drosophila.
Neuroscience. 21, 7705-7714.
Golemis, E.A., Gyuris, J. and Brent, R. (1994). Interaction trap/two hybrid system to
identify interacting proteins. In Current Protocols in Molecular Biology, pp. 13.14.1-
13.14.17. New York: Wiley.
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of
growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition
molecule. Cell 67, 45-57.
Guo, D., Tan, Y. C., Wang, D., Madhusoodanan, K. S., Zheng, Y., Maack, T., Zhang,
J. J. and Huang, X. Y. (2007). A Rac-cGMP signaling pathway. Cell 128, 341-355.
Guo, D., Zhang, J. J. and Huang, X. Y. (2010). A new Rac/PAK/GC/cGMP signaling
pathway. Mol. Cell. Biochem. 334, 99-103.
Hidalgo, A. and Brand, A. H. (1997). Targeted neuronal ablation: the role of pioneer
neurons in guidance and fasciculation in the CNS of Drosophila. Development 124,
Hirakawa, T., Galet, C., Kishi, M. and Ascoli, M. (2003). GIPC binds to the human
lutropin receptor (hLHR) through an unusual PDZ domain binding motif, and it
regulates the sorting of the internalized human choriogonadotropin and the density
of cell surface hLHR. J. Biol. Chem. 278, 49348-49357.
Hülsmeier, J., Pielage, J., Rickert, C., Technau, G. M., Klämbt, C. and Stork, T.
(2007). Distinct functions of alpha-Spectrin and beta-Spectrin during axonal
pathfinding. Development 134, 713-722.
Jeanneteau, F., Guillin, O., Diaz, J., Griffon, N. and Sokoloff, P. (2004). GIPC
recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling. Mol. Biol. Cell
Jeong, S., Juhaszova, K. and Kolodkin, A. L. (2012). The Control of semaphorin-1a-
mediated reverse signaling by opposing pebble and RhoGAPp190 functions in
drosophila. Neuron 76, 721-734.
Kedlaya, R. H., Bhat, K. M., Mitchell, J., Darnell, S. J. and Setaluri, V. (2006). TRP1
interacting PDZ-domain protein GIPC forms oligomers and is localized to
intracellular vesicles in human melanocytes. Arch. Biochem. Biophys. 454, 160-169.
Kim, J., Lee, S., Ko, S. and Kim-Ha, J. (2010). dGIPC is required for the locomotive
activity and longevity in Drosophila. Biochem. Biophys. Res. Commun. 402, 565-
Klaes A, Menne T, Stollewerk A, Scholz H, Klämbt C. (1994). The Ets transcription
factors encoded by the Drosophila gene pointed direct glial cell differentiation in the
embryonic CNS. Cell 78, 149-160.
Kolodkin, A. L. and Tessier-Lavigne, M. (2011). Mechanisms and molecules of
neuronal wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, a001727.
Landgraf, M., Bossing, T., Technau, G. M. and Bate, M. (1997). The origin, location,
and projections of the embryonic abdominal motorneurons of Drosophila.
Neuroscience. 17, 9642-9655.
Lin, D. M., Fetter, R. D., Kopczynski, C., Grenningloh, G. and Goodman, C. S.
(1994). Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation,
and altered fasciculation. Neuron 13, 1055-1069.
RESEARCH ARTICLEDevelopment (2014) doi:10.1242/dev.095968
Lou, X., Yano, H., Lee, F., Chao, M. V. and Farquhar, M. G. (2001). GIPC and GAIP
form a complex with TrkA: a putative link between G protein and receptor tyrosine
kinase pathways. Mol. Biol. Cell 12, 615-627.
Mahr, A. and Aberle, H. (2006). The expression pattern of the Drosophila vesicular
glutamate transporter: a marker protein for motoneurons and glutamatergic centers
in the brain. Gene Expr. Patterns 6, 299-309.
Ming, G. L., Song, H. J., Berninger, B., Holt, C. E., Tessier-Lavigne, M. and Poo, M.
M. (1997). cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225-
Mlodzik, M., Baker, N. E. and Rubin, G. M. (1990). Isolation and expression of
scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev. 4, 1848-1861.
Naccache, S. N., Hasson, T. and Horowitz, A. (2006). Binding of internalized
receptors to the PDZ domain of GIPC/synectin recruits myosin VI to endocytic
vesicles. Proc. Natl. Acad. Sci. USA 103, 12735-12740.
Nishiyama, M., Hoshino, A., Tsai, L., Henley, J. R., Goshima, Y., Tessier-Lavigne,
M., Poo, M. M. and Hong, K. (2003). Cyclic AMP/GMP-dependent modulation of
Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 423, 990-995.
Polleux, F., Morrow, T. and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant
for cortical apical dendrites. Nature 404, 567-573.
Potter, L. R. (2011). Guanylyl cyclase structure, function and regulation. Cell. Signal.
Ramadan, N., Flockhart, I., Booker, M., Perrimon, N. and Mathey-Prevot, B. (2007).
Design and implementation of high-throughput RNAi screens in cultured Drosophila
cells. Nat. Protoc. 2, 2245-2264.
Ranganayakulu, G., Schulz, R. A. and Olson, E. N. (1996). Wingless signaling
induces nautilus expression in the ventral mesoderm of the Drosophila embryo. Dev.
Biol. 176, 143-148.
Ruiz-Gómez, M., Coutts, N., Price, A., Taylor, M. V. and Bate, M. (2000). Drosophila
dumbfounded: a myoblast attractant essential for fusion. Cell 102, 189-198.
Schmidt, H., Stonkute, A., Jüttner, R., Koesling, D., Friebe, A. and Rathjen, F. G.
(2009). C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons.
Proc. Natl. Acad. Sci. USA 106, 16847-16852.
Schneider, I. (1972). Cell lines derived from late embryonic stages of Drosophila
melanogaster. J. Embryol. Exp. Morphol. 27, 353-365.
Seidel, C. and Bicker, G. (2000). Nitric oxide and cGMP influence axonogenesis of
antennal pioneer neurons. Development 127, 4541-4549.
Sepp, K. J., Schulte, J. and Auld, V. J. (2001). Peripheral glia direct axon guidance
across the CNS/PNS transition zone. Dev. Biol. 238, 47-63.
Shelly, M., Lim, B. K., Cancedda, L., Heilshorn, S. C., Gao, H. and Poo, M. M.
(2010). Local and long-range reciprocal regulation of cAMP and cGMP in
axon/dendrite formation. Science 327, 547-552.
Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne, M. and
Poo, M. (1998). Conversion of neuronal growth cone responses from repulsion to
attraction by cyclic nucleotides. Science 281, 1515-1518.
Tan, C., Deardorff, M. A., Saint-Jeannet, J. P., Yang, J., Arzoumanian, A. and
Klein, P. S. (2001). Kermit, a frizzled interacting protein, regulates frizzled 3
signaling in neural crest development. Development 128, 3665-3674.
Terman, J. R., Mao, T., Pasterkamp, R. J., Yu, H. H. and Kolodkin, A. L. (2002).
MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-
mediated axonal repulsion. Cell 109, 887-900.
Thompson, D. K. and Garbers, D. L. (1995). Dominant negative mutations of the
guanylyl cyclase-A receptor. Extracellular domain deletion and catalytic domain point
mutations. J. Biol. Chem. 270, 425-430.
Togashi, K., von Schimmelmann, M. J., Nishiyama, M., Lim, C. S., Yoshida, N.,
Yun, B., Molday, R. S., Goshima, Y. and Hong, K. (2008). Cyclic GMP-gated CNG
channels function in Sema3A-induced growth cone repulsion. Neuron 58, 694-707.
Van Vactor, D., Sink, H., Fambrough, D., Tsoo, R. and Goodman, C. S. (1993).
Genes that control neuromuscular specificity in Drosophila. Cell 73, 1137-1153.
Varsano, T., Dong, M. Q., Niesman, I., Gacula, H., Lou, X., Ma, T., Testa, J. R.,
Yates, J. R., III and Farquhar, M. G. (2006). GIPC is recruited by APPL to
peripheral TrkA endosomes and regulates TrkA trafficking and signaling. Mol. Cell.
Biol. 26, 8942-8952.
Wang, L. H., Kalb, R. G. and Strittmatter, S. M. (1999). A PDZ protein regulates the
distribution of the transmembrane semaphorin, M-SemF. J. Biol. Chem. 274, 14137-
Wills, Z., Bateman, J., Korey, C. A., Comer, A. and Van Vactor, D. (1999). The
tyrosine kinase Abl and its substrate enabled collaborate with the receptor
phosphatase Dlar to control motor axon guidance. Neuron 22, 301-312.
Winberg, M. L., Mitchell, K. J. and Goodman, C. S. (1998a). Genetic analysis of the
mechanisms controlling target selection: complementary and combinatorial functions
of netrins, semaphorins, and IgCAMs. Cell 93, 581-591.
Winberg, M. L., Noordermeer, J. N., Tamagnone, L., Comoglio, P. M., Spriggs, M.
K., Tessier-Lavigne, M. and Goodman, C. S. (1998b). Plexin A is a neuronal
semaphorin receptor that controls axon guidance. Cell 95, 903-916.
Yanagawa, S., Lee, J. S. and Ishimoto, A. (1998). Identification and characterization
of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J.
Biol. Chem. 273, 32353-32359.
Yi, Z., Petralia, R. S., Fu, Z., Swanwick, C. C., Wang, Y. X., Prybylowski, K., Sans,
N., Vicini, S. and Wenthold, R. J. (2007). The role of the PDZ protein GIPC in
regulating NMDA receptor trafficking. Neuroscience 27, 11663-11675.
Yu, H. H., Araj, H. H., Ralls, S. A. and Kolodkin, A. L. (1998). The transmembrane
Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon
guidance. Neuron 20, 207-220.
Yu, H. H., Huang, A. S. and Kolodkin, A. L. (2000). Semaphorin-1a acts in concert
with the cell adhesion molecules fasciclin II and connectin to regulate axon
fasciculation in Drosophila. Genetics 156, 723-731.
Zhao, Z. and Ma, L. (2009). Regulation of axonal development by natriuretic peptide
hormones. Proc. Natl. Acad. Sci. USA 106, 18016-18021.