Frazzled/DCC facilitates cardiac cell outgrowth and attachment during
Drosophila dorsal vessel formation
Frank D. Macabentaa,b, Amber G. Jensena,b, Yi-Shan Chenga, Joseph J. Kramera,
Sunita G. Kramera,b,n
aDepartment of Pathology and Laboratory Medicine Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes
Lane, Piscataway, NJ 08854, USA
bGraduate Program in Cell and Developmental Biology, UMDNJ-Graduate School of Biomedical Sciences & Rutgers, The State University of New Jersey, 190
Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
a r t i c l e i n f o
Received 12 December 2012
Received in revised form
29 April 2013
Accepted 7 May 2013
Available online 16 May 2013
a b s t r a c t
Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the
migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process
requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate
the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous
studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate
site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the
concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive
proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC
(Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and
that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in
the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell
shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is
impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB)
results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra
and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired
CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance
mechanisms contribute to lumen formation by preventing luminal domains from coming together,
site-specific Netrin/Frazzled signaling mediates CB attachment.
& 2013 Elsevier Inc. All rights reserved.
Formation of the Drosophila dorsal vessel (DV) requires a highly
stereotyped set of morphogenetic movements. During Drosophila
cardiac morphogenesis, 104 cardioblasts (CB) are specified in two
bilateral rows of cells that coordinately migrate towards the dorsal
midline where they undergo cell polarity and shape changes, and
make specific contacts across the dorsal midline to form the DV,
a single cell-layer linear tube with a central lumen. The rows
of CBs are flanked by two rows of non-muscle pericardial cells
(PCs) shown to coordinate dorsal migration with the overlying
ectoderm (Chartier et al., 2002). Similar to the primitive heart tube
in vertebrates, the Drosophila DV possesses anteroposterior polarity
and is subdivided, through the action of homeotic genes, into a
narrower anterior portion called the aorta and a wider more
posterior heart (Lo et al., 2002) (Fig. 1A). Within segments A2–A8,
CBs can be subdivided into either smaller contractile cells that
express the homeodomain gene tinman, or larger rounded cells
expressing the orphan nuclear receptor seven-up (Gajewski et al.,
2000). In the late stage embryonic DV, wingless (wg) expression
in three segmentally repeated double pairs of CBs marks the
differentiation of the seven-up positive CBs within the heart into
the inflow tracts called ostia (Lo et al., 2002; Molina and Cripps,
The proper morphogenesis of the heart depends on the precise
alignment of the two rows of CBs with each other at the dorsal
midline. As the rows approach one another, each CB undergoes a
series of dramatic cell shape changes and regulates its adhesive
interactions with the opposing cell in such a way that they become
attached at the dorsal-most and ventral-most points while main-
taining a space in between (see Fig. 5). Cell contact and adhesion
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0012-1606/$-see front matter & 2013 Elsevier Inc. All rights reserved.
nCorresponding author at: University of Medicine and Dentistry of New Jersey,
Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical
School, 675 Hoes Lane West, Piscataway, NJ 08854, USA. Fax: +1 732 235 4825.
E-mail address: email@example.com (S.G. Kramer).
Developmental Biology 380 (2013) 233–242
is first initiated dorsally, as each CB extends a leading edge
towards its contralateral counterpart across the dorsal midline,
resulting in an adhesive interaction. Subsequently, the CB extends
a second leading edge to make a ventral connection with its
counterpart. In this way, a sealed linear tube with a central lumen
is formed (Medioni et al., 2008; Santiago-Martinez et al., 2008).
Guidance molecules and adhesion proteins have been shown to
play a crucial role in the process of lumen formation. It was
previously shown that in embryos mutant for the gene shotgun,
which encodes Drosophila E-Cadherin (E-Cad), the adhesion
between opposing CBs is defective resulting in the absence of
lumen formation (Haag et al., 1999). Furthermore, our previous
findings show that E-Cad localizes specifically to the dorsal and
ventral attachment points and that this discrete localization is
dependent upon negative regulation of E-Cad by the Slit/Robo
pathway (Santiago-Martinez et al., 2008). In the absence of Slit/
Robo signaling, E-Cad is no longer restricted to the dorsal and
ventral attachment points and is found along the entire luminal
domain. As a result, the CBs become fully attached, leading to a
loss of luminal space (Medioni et al., 2008; Santiago-Martinez
et al., 2008). Similarly, the Unc5 receptor, which mediates a
repulsive response to the Netrin guidance cue, also participates
in lumen formation by providing repulsion of opposing CB
membranes in a Slit/Robo-independent manner (Albrecht et al.,
2011). However, while it is clear that repulsive guidance plays a
role in establishing the luminal space between opposing CBs, it is
still unknown what signal or signals are responsible for mediating
CB outgrowth, extension and attachment.
Netrin was originally identified as a diffusible long-range
Laminin-like extracellular axon guidance cue in Caenorhabditis
elegans (Ishii et al., 1992). Subsequently, Netrins have been found
in a wide range of animal species and have been shown to mediate
guidance and cell adhesion in many different cell types (reviewed
in (Lai Wing Sun et al., 2011)). Receptors for Netrins include the
single-pass transmembrane proteins Deleted in Colorectal Cancer
(DCC) and Unc5 (for review see (Lai Wing Sun et al., 2011).
Different receptor combinations have been shown to elicit attrac-
tive or repulsive responses to Netrin via signaling pathways that
ultimately result in the rearrangement of the actin cytoskeleton
(Moore et al., 2007). In general, DCC mediates chemoattraction,
while chemorepulsion requires Unc5 and in some cases DCC as
well. More recently, the Down Syndrome Cell Adhesion Molecule
(DSCAM) was also shown to function as both an attractive and
repulsive Netrin receptor in Drosophila and vertebrates (Andrews
et al., 2008; Ly et al., 2008; Purohit et al., 2012).
In Drosophila, DCC is called Frazzled (Fra) and was originally
indentified as a receptor for the attractive Netrin guidance cue in
the CNS (Harris et al., 1996; Kolodziej et al., 1996; Mitchell et al.,
1996). DCC/Fra has been shown to mediate chemoattractive
responses to Netrin in a variety of cell types (for a recent review
see (Lai Wing Sun et al., 2011)). Additionally, more recent studies
have shown that the DCC/Fra receptor becomes polarized on the
cell membrane to the areas corresponding to asymmetric cell
outgrowth and protrusion in a Netrin-dependent manner (Quinn
and Wadsworth, 2008). For example, the polarized protrusive
activity of the C. elegans HSN neuron is mediated by UNC-40 (C.
elegans DCC) in response to a ventrally localized Netrin signal
(Adler et al., 2006). Similarly, during C. elegans anchor cell
invasion, localized Netrin secretion directs UNC-40 to a specific
region of the anchor cell plasma membrane (Ziel et al., 2009).
However, the precise role for Netrin in polarizing DCC/Fra is
unclear, as recent evidence suggests that UNC-40 also has the
ability to intrinsically polarize in the absence of UNC-6 (C. elegans
Netrin) (Xu et al., 2009).
In this study, we show that the attractive Netrin receptor Fra is
involved in mediating asymmetric CB outgrowth and attachment
during DV morphogenesis. fra is expressed by the CBs, and genetic
perturbation of fra levels results in a failure of CBs to properly
come together at dorsal and/or ventral attachment points. Similar
defects are observed in embryos mutant for Netrin or fra, unc5
double mutants. Furthermore, we show that Fra accumulates at
the sites of CB outgrowth and attachment and that this accumula-
tion is correlated with proper lumen formation. Finally we show
that Fra localization at sites of attachment is disrupted in embryos
mutant for Netrin. Our findings provide evidence that just as
repulsive guidance via the Robo and Unc5 receptors is important
for preventing specific areas of the CB membranes from coming
into contact, so too is attractive guidance via the Fra receptor for
bringing together CBs at specific sites of adhesion.
Materials and methods
Fly stocks and genetics
Fly crosses were performed at 25 1C and maintained on standard
medium. The following stocks were used: (fra3) (BL#8813) and unc5
(unc58) (Labrador et al., 2005), ΔNetAB (Brankatschk and Dickson,
(G. Bashaw), 24B-Gal4 (Brand and Perrimon, 1993), Hand-Gal4 (Han
et al., 2006), UAS-mCD8-GFP (BL#5130). To generate fra rescue
embryos, fra3/CyO, twi-2XGFP; UAS-fra-myc flies were crossed to
fra3/CyO, twi-2XGFP; 24B-Gal4 or fra3, Hand-Gal4/CyO, twi-2xGFP flies.
For examining Fra-Myc localization in ΔNetAB mutants, FM7 sqh::
ChRFP; UAS-fra-myc males were crossed to ΔNetAB/FM7-Kr-Gal4,
UAS-GFP;24B-Gal4 females. For unc5 dominant suppression experi-
ments, UAS-fra-myc flies were crossed to unc58/Cyo,KrGFP; 24B-Gal4/
In situ hybridization
In situ hybridization on wild type and Hand-Gal4/UAS-lacZ
embryos was performed as described (Lecuyer et al., 2008). For
the fra antisense probe, the cDNA was linearized with BglII and
transcribed using DIG RNA labeling mix (Roche Diagnostics). The
Tyramide signal amplification kit was purchased from Molecular
Probes. Double labeling was performed with a Rabbit anti-βgal
antibody and a goat anti-Rabbit Alexa 555 secondary antibody.
Embryos were collected from grape-juice agar plates, dechor-
ionated and fixed according to standard protocols. The following
primary antibodies were used: rabbit anti-Mef2 (B. Paterson,
1:1000), rabbit anti-Myc (Sigma, 1:200), mouse anti-α-Spectrin
[Developmental Studies Hybridoma Bank (DSHB), 1:10], mouse
anti-Wingless (DSHB, 1:10), mouse anti-Myc (DSHB, 1:10), mouse
anti-Discs large (DSHB, 1:10), rabbit anti-β-gal (1:1000, MP Bio-
medicals), and rabbit anti-GFP (1:500, Invitrogen). For secondaries,
goat anti-mouse or anti-rabbit conjugated to either Alexa 488 or
555 (1:500; Invitrogen) were used. Fixed and stained embryos
were carefully staged using head and gut morphology and indivi-
dually mounted on glass coverslips in 60% glycerol. Confocal z-
sections were obtained at ambient temperature on an inverted
Olympus IX81 with a Crest CARV II confocal unit using a Plan
VApo/340 60X/1.20 NA W objective and an ORCA-EM CCD Digital
Well slides for viewing embryos in cross section were prepared
by painting small circles of valve lubricant (Dow Corning) on
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
24?60 mm2rectangular coverslips. A solution of heptane and
adhesive tape glue was applied in two layers, allowing the heptane
to evaporate between applications. Stage 16 or 17 embryos were
carefully staged using head and gut morphology and selected for
dissection on a separate slide. Using a scalpel, a transverse cut was
made through the entire diameter of each embryo approximately
two-thirds of the way from the most anterior end; the section
containing the most posterior end was then discarded. With the
aid of a needle, each dissected embryo was propped up vertically
on the center of the well slide. A solution of 60% glycerol diluted in
PBS was added drop-wise into the well, such that all embryos
were completely immersed. The embryos were then imaged using
an Olympus confocal microscope (described above). Statistical
analysis was performed using GraphPad Prism.
Transmission electron microscopy was performed as described
in (Soplop et al., 2009). Mutant embryos were selected based on
the absence of GFP balancer in live dechorionated embryos prior
Results and discussion
fra mRNA is expressed in the DV
fra encodes a transmembrane receptor for the Netrin guidance
cue (Harris et al., 1996; Kolodziej et al., 1996; Mitchell et al., 1996).
Previous studies have determined that fra is expressed in devel-
oping axons and epithelia in the Drosophila embryo ((Kolodziej
et al., 1996). However, fra expression in the DV has not yet been
reported. To determine whether fra mRNA was expressed in the
DV, we performed in situ hybridization to embryos using a fra
antisense probe. In addition to detecting the known expression of
fra in the CNS and epidermal tissues (Kolodziej et al., 1996; Fig. 1),
we also detected fra transcripts in the CBs as early as stage 14/15,
with the transcript persisting in CBs in stage 16 embryos (Fig. 1A
and B). We confirmed the CB expression of fra by double staining
β-galactosidase (β-gal) protein. Hand-Gal4 drives expression in
the CBs and a subset of the PCs (Han et al., 2006). Double staining
confirmed that fra mRNA accumulates in all the CBs (Fig. 1C and
D). We also detected fra mRNA in at least a subset of PCs (Fig. 1D).
Mutations in fra and Netrin cause DV defects
To investigate the potential role that fra plays during DV
development, we examined embryos mutant for the fra gene in
whole mount. In embryos homozygous for the molecularly char-
acterized EMS-induced amorphic fra3allele (Kolodziej et al., 1996),
we did not detect defects in the specification or overall number of
CBs, as revealed by staining with anti-DMef2 (Fig.1E and F), which
labels the nuclei of all 104 CBs. In addition, Pericardin, a collagen-
like protein that is secreted by the PCs and concentrates at the CB-
pericardial cell interface (Chartier et al., 2002), was properly
localized in fra3mutants (Fig. S1 B, C). While staining with anti-
Wg, which labels the six bilateral pairs of CB ostial cells in the
heart (Lo et al., 2002), showed that the ostial cells were correctly
specified, Wg staining did reveal significant defects in the dorsal
alignment of the two rows of CBs as seen by the shifting of the
contralateral pairs of Wg positive CBs relative to each other
(Fig. 1G and H). We observed this phenotype in 33% of fra3
homozygotes, which was significantly above what we observed
in wild type or fra3/+ embryos (Po0.05). The alignment pheno-
type, while mild, was also observed in the fra4allele (data not
shown) and is also consistent with a previous study in which the
authors isolated a new allele for fra in a genetic screen for heart
malformations (Meyer et al., 2011).
To further investigate the defects we observed in fra3mutants,
we stained fra3embryos with anti-αSpectrin (Lee et al., 1993),
which labels CB lateral and luminal membranes. αSpectrin-stain-
ing in fra3embryos confirmed the defects we observed in CB
alignment and also revealed defects in CB attachment at the dorsal
midline (Fig. 1J), without showing overall defects in CB polarity.
Specifically, in 26% (n¼35) of fra3mutant embryos observed in
whole mount, the dorsal-most membranes of opposing CBs did
not appear to be properly attached, and we could see a space
(spanning at least two or more cells) between opposing CBs
(Fig. 1K). This attachment phenotype was not detected in wild
type embryos. The defects in attachment did not appear to occur
predominantly in specific cell types (i.e. the Wg-expressing ostial
cells, which align with segment borders versus the sets of four
pairs of Tinman-expressing cardioblasts (Gajewski et al., 2000) in
between). In addition, we did not see gaps along the individual
rows, suggesting that this defect is not in the dorsal migration of
each row of CBs, but rather in the contact of CBs across the dorsal
midline. Double staining with anti-DMef2 confirmed that these
spaces were not due to the presence of extra CBs but most likely
resulted from an attachment failure between contralateral CBs
Fra is a known receptor for the secreted Netrin molecule, which
is encoded by two genes, NetrinA (NetA) and NetrinB (NetB) in
Drosophila (Harris et al., 1996; Kolodziej et al., 1996; Mitchell et al.,
1996). While NetB protein has been reported to localize to the CBs
(Albrecht et al., 2011; Harris et al., 1996), there are conflicting data
in the literature regarding the source of secreted NetB in the CBs.
While an early study reported weak NetB mRNA in the DV
(Mitchell et al., 1996), a more recent study reported a failure to
detect NetB transcript and suggested that NetB is diffusing from
another source (Albrecht et al., 2011). In order to determine if the
fra3alignment and attachment defects we observed are down-
stream of Netrin signaling, we examined ΔNetAB embryos, in
which both Netrin genes are specifically deleted by P-element
excision (Brankatschk and Dickson, 2006). Staining with anti-Wg
revealed occasional defects in CB alignment at the dorsal midline;
however, the defects were not significantly above wild type levels
(data not shown). Staining with anti-αSpectrin did reveal signifi-
cant defects in CB attachment at the dorsal midline. Specifically,
we found that 30% (n¼23) of ΔNetAB embryos showed defects in
CB attachment (Fig. 1M). These defects were similar to what we
observed in fra3mutants (Fig. 1K). Together, these results suggest
that Netrin and Fra function together during DV formation in
mediating CB attachment.
fra is required for CB attachment during DV morphogenesis
In order to more closely examine the roles of Fra and Netrin in
the DV, embryos were cross-sectioned, which allowed us to
visualize the specific contacts that are made by the CBs as well
as the size and shape of the lumen. During morphogenesis of the
DV, the rounded CBs undergo a series of cell shape changes,
enabling first the dorsal and then the ventral region of each CB
to initiate contact with their contralateral partner across the dorsal
midline, while the areas in between remain unattached allowing
for the formation of a lumen (Haag et al., 1999; Medioni et al.,
2008; Santiago-Martinez et al., 2008).
We first cross-sectioned both wild type and fra3embryos that
were immunostained for αSpectrin to visualize CB membranes
(see Materials and methods). In wild type embryos at early stage
16, the dorsal-most contact between contralateral CBs has been
established (Fig. 2A and A') and ventral contact is initiated.
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
By stage 17, the CBs are attached at both dorsal and ventral
attachment points and a lumen can be observed in between
(Fig. 2B and B'). In fra3mutants, we observed significant defects
(85%, n¼20) that arose from the failure of CBs to make appropriate
contacts with each other across the dorsal midline at stage 17
(Table 1). We observed two main classes of phenotypes that range
in severity. In the milder class, the CBs appeared to have under-
gone proper cell shape changes but then showed a missing or
diminished area of dorsal and/or ventral contact, leading to an
open-heart tube or a severely enlarged lumen (Fig. 2C and C').
In the second class, the CBs failed to undergo the cell shape
changes required for CB outgrowth and as a result did not make
either dorsal or ventral contact with each other across the dorsal
midline (Fig. 2D, D'). In these sections, a clear space could be
observed between the opposing CB membranes and we presume
that this is the attachment phenotype that is clearly visible in our
whole mount embryos (Fig. 1K and L). We confirmed that these
embryos were not of an earlier stage or generally delayed in
Fig. 1. fra is expressed in the dorsal vessel and fra mutants have defects in CB alignment and attachment. Top panel shows a schematic of the embryonic DV, which spans
segments T2–A8 and is divided into the anterior aorta, posterior aorta and the heart. In the heart, the six pairs of Wingless-expressing CB ostial cells are indicated in magenta.
(A, B) In situ hybridization was performed with a DIG-labeled fra antisense probe and visualized using Tyramide signal amplification. Expression of fra at stage 15 is highly
enriched in the DV (A). fra expression persists through stage 16 (B). (C, D) Co-staining Hand-Gal4/UAS-lacZ embryos for fra mRNA (green) and β-gal (red) shows the expression
of fra mRNA on both the CBs and PCs (arrows). (E-F) anti-DMef2 staining in fra3mutants at stage 14 (E) and 16 (F) showing that CB specification and migration to the dorsal
midline is not impaired in fra3mutants. Wild type embryos have 104 CBs, and we found no significant difference in CB number in fra3mutants (104+/−2, n¼5). (G-H)
Staining with anti-Wingless (Wg) in wild type (wt) (G) or fra3mutant (H) reveals that the ostial cells (brackets) are correctly specified in fra3mutants, but are often mis-
aligned across the dorsal midline (asterisks). (I-J) anti-αSpectrin staining in the heart region of the DV. In wild type stage 16 embryos, the ostial cells (brackets) are aligned
with each other and make contact across the dorsal midline (I). In fra3mutants (J), ostial cells (brackets) are often mis-aligned (asterisk) and fail to make proper contact with
their contralateral counterparts (arrows). (K) Another fra3mutant embryo showing mis-aligned CB ostial cells (brackets) and failure of CBs to make contact across the dorsal
midline (arrow). (L) fra3embryo double stained with anti-DMef2 (red) and anti-αSpectrin (green) to show that the space between CBs is not due to the presence of an extra
CB. (M) ΔNetAB mutant embryos also show defects in CB attachment (arrows).
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
development by simultaneously examining head involution, gut
morphology and dorsal closure with αSpectrin, which not only
labels the CB membranes but also epidermal cells. We observed
what appeared to be mild attachment defects in 31% of wild type
embryos (Table 1), potentially reflecting that lumen formation
does not simultaneously occur along the length of the embryo and
that there are occasionally some CB pairs that have not completed
the process at stage 17. However at this late stage in wild type
embryos, we never observed a complete failure of CB outgrowth
and the large space between contralateral CBs that we observed in
fra mutants (Fig. 2D).
NetAB mutants share CB attachment phenotypes with fra mutants
In ΔNetAB mutants, the dominant phenotype was in CB attach-
ment, similar to what we observed in fra mutants. Specifically, in
74% of the embryos we examined (n¼19) (Table 1), contralateral
CBs had either diminished attachment domains leading to an
enlarged lumen (Fig. 2F and F') or a complete failure to attach
(Fig. 2G and G'). We also observed a small number of embryos (5%)
that showed CBs inappropriately attached along their entire
luminal domain, resulting in a loss of lumen formation (Table 1).
This phenotype was never observed in wild type embryos
(Table 1), and is identical to the predominant phenotype we
observed in unc5 loss-of-function embryos (36%, n¼25) (Fig. 2I
and I') (Table 1). This loss of lumen phenotype we observed in unc5
mutants as well as a small subset of Netrin mutants is consistent
with the phenotypes reported in a recent study, which examined
the role of unc5 during DV lumen formation (Albrecht et al., 2011).
Together, these data demonstrate that Fra and Netrin function in
mediating CB outgrowth and attachment and suggest, based on
Fig. 2. frazzled and Netrin mutants have defects in CB attachment. (A–I) Cross-sections taken through the heart region of the DV (abdominal segments A6–A8) of embryos
immunostained with anti-αSpectrin, which outlines the CB membranes. Variances in αSpectrin staining between panels do not necessarily reflect differences in αSpectrin
localization but rather in image processing. Except for panel (A), all embryos shown are at stage 17. (A'–I') are representative cartoons showing the shape of the CBs based on
the cross-sections in the corresponding figures. (A, B) wild type (wt) embryos. (A) In early stage 16 wt embryos, contralateral CBs initiate contact at their dorsal-most regions
(arrow). (B) By stage 17, both dorsal and ventral contacts have formed (arrows), and a lumen can be observed in between. (C). fra3mutants show a range of lumen defects
(C and D). (C) Diminished adhesion domain phenotype where dorsal and ventral contacts appear to form (arrowheads) but are severely diminished compared to wt. In
addition, the lumen appears enlarged. (D) Arrested phenotype, where CBs migrate to the dorsal midline, but then fail to undergo further cell shape changes resulting in a
large space in between contralateral CBs (arrow). (E) Overexpression of UAS-fra at high levels in CBs with 24B-Gal4 results in an “arrest” phenotype, where the CBs have failed
to initiate dorsal contact (arrow). (F, G) In ΔNetAB embryos, the attachment points between CBs are often diminished or absent (arrows) (F) or CBs fail to undergo cell shape
changes leading to failure to make contact (arrow) (G). (H) unc5, fra double mutants show an “arrest” phenotype in which the CBs fail to make contact. (I) In unc58mutants,
we observed inappropriate attachment of CBs (arrow) resulting in a no-lumen phenotype.
Quantification of CB phenotypes in cross-sectioned embryos.
No defect Defect in CB–CB contact No lumen
fra3; UAS-fra X Hand-Gal4
fra3; UAS-fra X 24B-Gal4
UAS-fra X Hand-Gal4
UAS-fra X 24B-Gal4
unc58/+; UAS-fra X 24B-Gal4 65% (13/20) 35% (7/20)
69% (20/29) 31% (9/29)
74% (17/23) 26% (6/23)
aData sets differ significantly from WT with a P value of o0.05 by the T-test.
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
the phenotypes we observed, that this function may occur inde-
pendently of Unc5-mediated CB repulsion.
fra and unc5 double mutants look like fra mutants
unc5 mutants fail to form a lumen due to a loss of repulsion
leading to inappropriate contact between contralateral CBs
(Albrecht et al., 2011; Fig. 2I and I'). This is in contrast to fra
mutants in which there is a failure of contralateral CBs to make
appropriate contact, presumably due to a loss of attraction (Fig. 2C
and D). In order to determine the relative roles of these two
opposing Netrin receptors, we examined unc5 and fra double-
mutants and found that the phenotype of the double mutant much
more closely resembles the fra single-mutant phenotype (Fig. 2H,
H'; Table 1) in which contralateral CBs fail to make contact at the
dorsal midline. These data are consistent with the idea that fra
functions prior to and independently of unc5 during lumen
formation. This is further supported by our observation that Netrin
mutants also predominantly display the fra mutant phenotype
(Fig. 2F and G) and is consistent with previous studies showing
that contralateral CBs must first initiate dorsal contacts before
undergoing the cell shape changes leading to formation of the
lumen (Haag et al., 1999; Medioni et al., 2008; Santiago-Martinez
et al., 2008).
fra mutants show a loss of CB adhesion
Our results thus far are consistent with the idea that Fra is
playing an important role in bringing together the CBs at specific
sites of cell–cell contact. To more directly examine if cell contact
and adhesion were specifically disrupted in fra mutants, we first
performed EM analysis. Our EM data confirmed our analysis by
cross-section. In fra3mutants, we often observed an enlarged
luminal space between contralateral CBs, with the CB attachment
domains severely decreased as compared to wild type (Fig. 3A and
B). In contrast, unc5 mutants show CBs that are improperly
attached at the luminal domain, resulting in a loss of lumen
formation (Fig. 3C).
To further investigate whether fra mutants show a loss of CB cell
contact, fra3mutant embryos were stained for the adhesion and
junctional protein Discs-large (Dlg) (Woods et al., 1996). In fra3/+
heterozygous embryos viewed in cross-section, Dlg localizes to the
dorsal and ventral points of CB–CB contact as previously observed for
wild type embryos (Medioni et al., 2008; Vanderploeg et al., 2012)
(Fig. 3D). In fra3mutants, we see an absence of Dlg accumulation
between CBs, indicating that the junctional domains did not properly
form (Fig. 3E). In contrast, unc58mutants showed an inappropriate
accumulation of Dlg, consistent with the idea that CBs are adhered
along the entire luminal domain, resulting in the absence of lumen
formation (Fig. 3F).
Overexpression of fra leads to CB attachment defects
To further test the importance of fra in the DV, we performed a
series of rescue and gain-of-function studies by driving a UAS-fra
transgene in the DV with either the Hand-Gal4 or 24B-Gal4 drivers.
Both Hand-Gal4 and 24B-Gal4 drive expression in the DV (Brand
and Perrimon, 1993; Han et al., 2006). However, we found subtle
but important differences between these drivers. First, Hand-Gal4
appears to drive expression in the CBs at lower levels than 24B-
Gal4 (data not shown). Second, 24B-Gal4, which is known to
reflect the expression of the how gene (Zaffran et al., 1997), does
not drive expression uniformly in all CBs. For example, driving
UAS-mCD8-GFP (a membrane-tethered fusion protein between
mouse lymphocyte marker CD8 and the green fluorescence
protein, (Lee and Luo, 1999) with 24B-Gal4 resulted in the
accumulation of CD8-GFP at higher levels in the ostial CBs, which
are aligned with segment borders, as compared with the sets of
four pairs of contractile CBs in between (Fig. S2A).
We first attempted to rescue the fra mutant phenotype by
driving UAS-fra in fra mutant embryos with either 24B-Gal4 or
Hand-Gal4. We were not able to significantly rescue the loss of CB
attachment phenotype using either driver (Table 1). Furthermore,
while driving expression of UAS-fra with the weaker Hand-Gal4 in
an otherwise wild type background did not have an effect
(Table 1), overexpression of UAS-fra at higher levels with 24B-
Gal4 in wild type embryos resulted in significant CB attachment
phenotypes, similar to what we observed in fra loss-of-function
mutants (Table 1, Fig. 2E and E'). Interestingly, the fra gain-of-
function phenotypes we observed with the 24B-Gal4 driver
occurred at a much higher frequency between the ostial CBs,
which show much higher levels of Fra-Myc expression (Fig. S2B)
than the four pairs of contractile CBs in between (75% vs. 31%
respectively). We were able to clearly identify the ostial CB cells in
our cross-sections by their proximity to the alary muscles, which
connect to the DV to support the cardiac tube as well as control
hemolymph inflow (Bate, 1993; Rizki, 1978).
Together our results suggest that tight temporal and/or spatial
regulation of Fra receptor levels in CBs is essential for the initiation
and/or completion of the cell shape changes required for lumen
Fig. 3. fra mutants show diminished adherent domains. TEM sections through the heart of stage 17 wild type (wt) (A), fra3and unc58mutant embryos (B) and
(C) respectively. In fra3mutants (B) the area of CB–CB contact is diminished (arrows) and the lumen appears enlarged as compared with wt. In contrast, unc5 mutants (C) are
inappropriately adhered along the entire CB face, resulting in the absence of a lumen (arrow). In fra3/+ heterozygotes, Discs large (Dlg), a junctional marker is enriched at the
dorsal and ventral sites of CB contact (arrows) (D). In fra3/ fra3embryos, Dlg fails to accumulate at these points (arrows) (E). In unc58mutants, Dlg accumulates between CBs
that are inappropriately attached (F).
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
formation, and that neither the 24B-Gal4 nor Hand-Gal4 drivers
fully recapitulate normal levels of fra expression required for
rescue of the attachment phenotypes in fra mutants. In addition,
our overexpression data are consistent with findings in other
systems that suggest that Fra function is highly regulated and that
overexpression of fra often causes a disruption to its normal
function phenotypes (Levy-Strumpf and Culotti, 2007; Timofeev
et al., 2012; Watari-Goshima et al., 2007). An alternative inter-
pretation of these results is that when fra is expressed at high
levels in the CBs, it is able to function with Unc5 to mediate long-
range repulsion, thus preventing the CBs from coming together.
These data are supported by the findings that Unc5 functions as
both a short-range and long-range repellent, and that only long-
range repulsion requires Fra (Keleman and Dickson, 2001).
In order to test this possibility, we overexpressed fra with 24B-
Gal4 in embryos that were heterozygous for unc5. Interestingly, we
found that unc5 was able to dominantly suppress the fra gain-of-
function phenotype to near wild type levels (Table 1). These
results are consistent with the idea that when expressed at high
levels, fra can work in concert with unc5 to mediate long range
repulsion of CBs, thus preventing CB attachment.
loss-of-function and gain-of-
Fra localizes to sites of CB outgrowth and attachment
Our results thus far show that Fra functions in the CBs for
proper outgrowth and attachment. Earlier studies have shown that
the Fra homologs Unc-40 and DCC localize to distinct subcellular
locations on the cell membrane, often in response to Netrin
signaling (Adler et al., 2006; Matsumoto and Nagashima, 2010;
Ziel et al., 2009). While our findings clearly showed that fra mRNA
is expressed by the CBs (Fig. 1A–D), we were unable to detect
endogenous Fra protein above background levels using an anti-Fra
antibody previously used to localize the protein in the CNS (Garbe
and Bashaw, 2007). As an alternative method to determine
whether Fra protein has a specific subcellular distribution in the
CBs, we examined the expression of the UAS-fra-myc transgene
driven by either Hand-Gal4 or 24B-Gal4. In performing these
experiments, we took advantage of the differences between the
Hand and 24B-Gal4 drivers in our analysis. We first examined
Hand-Gal4/UAS-fra-myc embryos stained with anti-Myc. Overex-
pression of fra using the Hand-Gal4 driver did not result in
significant defects in CB attachment (Table 1). During the initial
stages of DV formation, as the CBs approached the midline, we
were unable to detect high levels of Fra-Myc accumulation in CBs
in these embryos (data not shown). However, at stage 17, when
lumen formation was complete, we observed the accumulation of
high levels of Fra-Myc at the sites of CB–CB contact (Fig. S3A).
To determine the localization of Fra in CBs at earlier stages, we
examined UAS-fra-myc expression driven by the stronger 24B-Gal4
driver. A potential complication of using this driver to localize Fra
is that overexpression of UAS-fra caused significant defects in CB
attachment (Table 1, Fig. 2E). Because our subsequent analysis
showed that these defects occurred much more frequently in the
ostial CBs (see above), we initially focused our analysis on sections
through the non-ostial CBs that showed a normal morphology. In
these sections, we noticed that as contralateral CBs approached
the dorsal midline at early stage 16, Fra-Myc staining became
enriched first at the CB dorsal leading edge (Fig. 4A and A') and
subsequently at the ventral leading edge (Fig. 4B and B'). Finally, at
the completion of lumen formation at stage 17, Fra-Myc staining
persisted at areas of CB contact (Fig. 4C and C'). As a control for our
localization experiments, we used 24B-Gal4 to drive the expres-
sion of UAS-mCD8-GFP, a heterologous membrane protein. We
found that unlike the highly localized pattern that we observed for
Fra-Myc, the mCD8-GFP protein appeared to be evenly distributed
along the surface of the CBs, as visualized with anti-GFP staining
(Fig. S3B). These results are consistent with the idea that Fra
localization specifically correlates with areas of CB membrane
outgrowth and attachment.
We next questioned whether the attachment phenotype that we
observed between CB ostial cells in fra-overexpressing embryos
could be correlated with changes in Fra-Myc localization. We
examined Fra-Myc localization in 24B-Gal4/UAS-fra-Myc embryos
double-stained with anti-Wg, a marker for the CB ostial cells (Lo
et al., 2002) and anti-Myc. We hypothesized that Wg-negative CBs
expressing lower levels of UAS-fra-Myc would show normal CB
morphology and by extension, the Fra-Myc staining would reflect a
normal pattern of Fra localization. In contrast, we hypothesized that,
higher levels of UAS-fra-Myc expression would lead to defects in CB
attachment as well as mislocalization of Fra-Myc. As expected, we
found that cross-sections through the high-fra-Myc expressing Wg
positive ostial CBs clearly showed a more homogeneous distribution
of Fra-Myc as well as significant CB attachment defects (Fig. 4D and
E). In contrast, cross-sections through non-Wg expressing cells
showed a more restricted distribution of Fra-Myc at sites of CB
attachment and a normal lumen (Fig. 4F). We further confirmed this
by examining additional cross-sections from a single embryo that we
double stained with anti-Myc and anti-αSpectrin, to label the entire
CB membrane (Fig. 4 G–L). In these sections, due to the lack of Wg
staining, we identified the higher Fra-Myc expressing CB ostial cells
by their proximity to the alary muscles. In sections taken through the
CB ostial cells, we observed clear defects in CB morphogenesis
together with a more homogeneous localization of Fra-Myc on the
CB membrane (Fig. 4 G–I). In contrast, a neighboring section taken
through a non-ostial CB shows accumulation of Fra-Myc at sites of CB
outgrowth and attachment (Fig. 4J–L).
Together, these results support the idea that Fra accumulation
at sites of CB outgrowth and adhesion is required for proper lumen
formation. Our findings are consistent with previous studies
showing that Fra/Unc-40 becomes polarized on cell membranes
that correspond to areas of cell outgrowth (Adler et al., 2006; Ziel
et al., 2009). However, it is important to note that all of our
observations are based on overexpression studies and therefore
may not entirely reflect the localization of endogenous Fra protein
in the DV.
Fra accumulation at sites of CB attachment is disrupted in Netrin
Netrins have previously been shown to promote the recruitment
of DCC/Fra to distinct locations of the cell membrane (Adler et al.,
2006; Matsumoto and Nagashima, 2010). Furthermore, in the
Drosophila CNS, Fra was shown to relocalize in response to Netrin
signaling (Hiramoto et al., 2000). In the DV, both fra and Netrin
mutants showed significant defects in CB attachment (Fig. 2 E and
G, Table 1), suggesting that they function together during this
process. To test whether the accumulation of Fra that we observed
at specific points of CB attachment was dependent upon Netrin, we
repeated our Fra-Myc localization studies described above in
embryos that were heterozygous or homozygous for ΔNetAB.
Because we observed Fra-Myc accumulation at sites of attachment
specifically in the non-ostial CBs, we focused on these cells for our
analysis. In ΔNetAB/+; 24B-Gal4/UAS-fra-Myc embryos, we detected
Fra-Myc at points of CB attachment (Fig. 4M), similar to the pattern
we saw in wild type embryos at stage 17 (Fig. 4C). However, in
embryos homozygous for ΔNetAB, we often found that Fra-Myc
staining was more diffusely distributed around the CB membrane or
inappropriately accumulated at areas not normally associated with
CB attachment (Fig. 4N). However, in some sections, we also
observed normal distribution of Fra-Myc (Fig. 4O). Together, these
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
findings support the idea that Fra localization at sites of attachment
may at least be partially dependent upon Netrin signaling.
In this paper we have shown an important role for the Fra
protein during DV formation. DV formation occurs via a series of
highly stereotyped cell shape changes resulting in a linear tube
comprising of two rows of CBs that are attached at their dorsal and
ventral-most points, with a lumen in between (Fig. 5). Cell contact
and adhesion is first initiated dorsally, as each CB extends a
leading edge towards its contralateral counterpart across the
dorsal midline, resulting in an adhesive interaction (Fig. 5B–C).
By using loss-of-function analysis, we show that fra is required for
this CB outgrowth. In addition, we show that Fra protein accumu-
lates at sites of CB outgrowth and attachment (Fig. 5B–D) and that
this localization of Fra can be correlated with proper CB morpho-
genesis. Overexpression of Fra at high levels results in a homo-
geneous distribution of Fra along the entire CB membrane leading
to defects in CB attachment. Furthermore, we show that embryos
mutant for both the fra and unc5 receptors or their common ligand
Fig. 4. Frazzled protein concentrates at sites of CB attachment (A–C) Cross-sections of embryos expressing UAS-fra-Myc in all CBs with 24B-Gal4. (A) Anti-Myc staining at
early stage 16 reveals that Fra-Myc accumulates dorsally in the CBs corresponding to the initial sites of contact (arrow). (B) At late stage 16, Fra-Myc also accumulates at the
site of ventral CB outgrowth (arrow). Dorsal contact is indicated with an asterisk. (C) Stage 17 embryo when lumen formation is complete. Fra-Myc staining persists at dorsal
and ventral sites of contact (asterisks). Schematic diagrams illustrating Fra-Myc localization (A'–C'). (D, F) Double labeling of UAS-fra-Myc/24B-Gal4 embryo with anti Myc and
anti-Wg, which labels the 6 pairs of ostial CBs in the heart. CBs that were Wg positive showed CB attachment phenotypes as well as uniform distribution of Fra-Myc (D and
E). In CBs that were negative for Wg protein (F), Fra-Myc accumulated at dorsal and ventral points of attachment (arrows) and the lumen appeared normal. (G-I) UAS-fra-myc/
24B-Gal4 embryo sectioned through the ostial CBs, which express high levels of Fra-Myc. In these embryos, Fra-Myc staining (G) was evenly distributed along the entire CB
membrane, similar to αSpectrin (H). (I) is a merge of (G) and (H). (J–L) UAS-fra-myc/24B-Gal4 embryo sectioned through the non-ostial CBs expressing lower levels of fra-Myc.
Arrows point to the areas that accumulate Fra-Myc protein. (L) is a merge of (J) and (K). (M–O) (M) Fra-Myc accumulates at dorsal and ventral attachment points (asterisks) in
a ΔNetAB heterozygote at stage 17. (N) In a ΔNetAB mutant, Fra-Myc accumulation at these sites is disrupted (arrows) and we observe inappropriate accumulation of Fra-Myc
on areas of the CB membrane not normally associated with attachment (arrowhead). In these sections a proper lumen fails to form. (O) In ΔNetAB mutants that show a
normal lumen, we also observe normal distribution of Fra-Myc (asterisks).
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
Netrin primarily display a fra phenotype, demonstrating that fra-
mediated CB outgrowth and attachment occurs prior to and
largely independent of unc5-mediated lumen formation. Interest-
ingly, the pattern of Fra is complementary to those reported for the
Unc5 and Robo receptors, which are localized to the CB lumen
where they are required for repulsion of CB membranes in order to
form a luminal space (Fig. 5;Albrecht et al., 2011; Santiago-
Martinez et al., 2008). We previously showed that overexpression
of Robo results in inappropriate expansion of the luminal domain
and a loss of CB–CB contact (Santiago-Martinez et al., 2008). Thus,
CB morphogenesis occurs by the sequential outgrowth and inhibi-
tion of discrete CB membrane domains. It is still unclear how these
opposing signaling pathways are asymmetrically regulated inside
the cell. Our findings together with the known localization
patterns of Unc5 and Robo suggest that the localization of these
receptors to discrete membrane domains is essential for proper CB
We thank: Greg Bashaw, Barry Dickson and B. Paterson, Zhe
Han for reagents, Rajesh Patel for assistance with EM, and
members of the Wadsworth lab for helpful discussions. We would
also like to acknowledge the Bloomington Stock Center at the
Indiana University for providing fly stocks, and the Developmental
Studies Hybridoma Bank developed under the auspices of the
NICHD and maintained by The University of Iowa, Department
of Biology for antibodies. This work was funded by NIH
R01AR054482 from NIAMS for S.G.K. and a Rutgers/UMDNJ Bio-
technology Training Grant T32 GM008339 for F.D.M.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2013.05.007.
Adler, C.E., Fetter, R.D., Bargmann, C.I., 2006. UNC-6/Netrin induces neuronal
asymmetry and defines the site of axon formation. Nat. Neurosci. 9, 511–518.
Albrecht, S., Altenhein, B., Paululat, A., 2011. The transmembrane receptor Uncoor-
dinated5 (Unc5) is essential for heart lumen formation in Drosophila melano-
gaster. Dev. Biol. 350, 89–100.
Andrews, G.L., Tanglao, S., Farmer, W.T., Morin, S., Brotman, S., Berberoglu, M.A.,
Price, H., Fernandez, G.C., Mastick, G.S., Charron, F., Kidd, T., 2008. Dscam guides
embryonic axons by Netrin-dependent and -independent functions. Develop-
ment 135, 3839–3848.
Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell
fates and generating dominant phenotypes. Development 118, 401–415.
Brankatschk, M., Dickson, B.J., 2006. Netrins guide Drosophila commissural axons at
short range. Nat. Neurosci. 9, 188–194.
Chartier, A., Zaffran, S., Astier, M., Semeriva, M., Gratecos, D., 2002. Pericardin, a
Drosophila type IV collagen-like protein is involved in the morphogenesis and
maintenance of the heart epithelium during dorsal ectoderm closure. Devel-
opment 129, 3241–3253.
Gajewski, K., Choi, C.Y., Kim, Y., Schulz, R.A., 2000. Genetically distinct cardial cells
within the Drosophila heart. Genesis 28, 36–43.
Garbe, D.S., Bashaw, G.J., 2007. Independent functions of Slit-Robo repulsion and
Netrin-Frazzled attraction regulate axon crossing at the midline in Drosophila.
J. Neurosci. 27, 3584–3592.
Haag, T.A., Haag, N.P., Lekven, A.C., Hartenstein, V., 1999. The role of cell adhesion
molecules in Drosophila heart morphogenesis: faint sausage, shotgun/DE-
cadherin, and laminin A are required for discrete stages in heart development.
Dev. Biol. 208, 56–69.
Han, Z., Yi, P., Li, X., Olson, E.N., 2006. Hand, an evolutionarily conserved bHLH
transcription factor required for Drosophila cardiogenesis and hematopoiesis.
Development 133, 1175–1182.
Harris, R., Sabatelli, L.M., Seeger, M.A., 1996. Guidance cues at the Drosophila CNS
midline: identification and characterization of two Drosophila Netrin/UNC-6
homologs. Neuron 17, 217–228.
Hiramoto, M., Hiromi, Y., Giniger, E., Hotta, Y., 2000. The Drosophila Netrin receptor
Frazzled guides axons by controlling Netrin distribution. Nature 406, 886–889.
Ishii, N., Wadsworth, W.G., Stern, B.D., Culotti, J.G., Hedgecock, E.M., 1992. UNC-6, a
laminin-related protein, guides cell and pioneer axon migrations in C. elegans.
Neuron 9, 873–881.
Keleman, K., Dickson, B.J., 2001. Short- and long-range repulsion by the Drosophila
Unc5 netrin receptor. Neuron 32, 605–617.
Kolodziej, P.A., Timpe, L.C., Mitchell, K.J., Fried, S.R., Goodman, C.S., Jan, L.Y., Jan, Y.N.,
1996. frazzled encodes a Drosophila member of the DCC immunoglobulin
subfamily and is required for CNS and motor axon guidance. Cell 87, 197–204.
Labrador, J.P., O’Keefe, D., Yoshikawa, S., McKinnon, R.D., Thomas, J.B., Bashaw, G.J.,
2005. The homeobox transcription factor even-skipped regulates netrin-
receptor expression to control dorsal motor-axon projections in Drosophila.
Curr. Biol. 15, 1413–1419.
Fig. 5. Summary of the roles of Frazzled, Unc-5 and Slit/Robo during dorsal vessel
morphogenesis. Schematic showing four stages of dorsal vessel morphogenesis as
visualized in cross-section. Panel (A) shows contralateral cardioblasts (CBs, red) as
they are migrating towards the dorsal midline. (B) As they approach the midline,
contralateral CBs extend their dorsal-most membrane regions. These membrane
domains accumulate Frazzled protein (blue). (C) Following contact of the dorsal
membranes, CBs extend their Frazzled-rich ventral membranes, while the regions
in-between remain unattached due to Slit/Robo (orange) and Unc-5 (yellow)
signaling. (D) Closure of the dorsal vessel showing Frazzled protein persisting at
dorsal and ventral contact points. (For interpretation of the references to color in
this figure legend, the reader is reffered to the web version of this article.)
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242
Lai Wing Sun, K., Correia, J.P., Kennedy, T.E., 2011. Netrins: versatile extracellular
cues with diverse functions. Development 138, 2153–2169.
Lecuyer, E., Necakov, A.S., Caceres, L., Krause, H.M., 2008. High-resolution fluor-
escent in situ hybridization of Drosophila embryos and tissues. Cold Spring
Harb. Protoc., http://dx.doi.org/10.1101/pdb.prot5019.
Lee, J.K., Coyne, R.S., Dubreuil, R.R., Goldstein, L.S., Branton, D., 1993. Cell shape and
interaction defects in alpha-spectrin mutants of Drosophila melanogaster. J. Cell
Biol. 123, 1797–1809.
Lee, T., Luo, L., 1999. Mosaic analysis with a repressible cell marker for studies of
gene function in neuronal morphogenesis. Neuron 22, 451–461.
Levy-Strumpf, N., Culotti, J.G., 2007. VAB-8, UNC-73 and MIG-2 regulate axon
polarity and cell migration functions of UNC-40 in C. elegans. Nat. Neurosci. 10,
Lo, P.C., Skeath, J.B., Gajewski, K., Schulz, R.A., Frasch, M., 2002. Homeotic genes
autonomously specify the anteroposterior subdivision of the Drosophila dorsal
vessel into aorta and heart. Dev. Biol. 251, 307–319.
Ly, A., Nikolaev, A., Suresh, G., Zheng, Y., Tessier-Lavigne, M., Stein, E., 2008. DSCAM
is a netrin receptor that collaborates with DCC in mediating turning responses
to netrin-1. Cell 133, 1241–1254.
Matsumoto, H., Nagashima, M., 2010. Netrin-1 elevates the level and induces
cluster formation of its receptor DCC at the surface of cortical axon shafts in an
exocytosis-dependent manner. Neurosci. Res. 67, 99–107.
Medioni, C., Astier, M., Zmojdzian, M., Jagla, K., Semeriva, M., 2008. Genetic control
of cell morphogenesis during Drosophila melanogaster cardiac tube formation.
J. Cell Biol. 182, 249–261.
Meyer, H., Panz, M., Albrecht, S., Drechsler, M., Wang, S., Husken, M., Lehmacher, C.,
Paululat, A., 2011. Drosophila metalloproteases in development and differentia-
tion: the role of ADAM proteins and their relatives. Eur. J. Cell Biol. 90, 770–778.
Mitchell, K.J., Doyle, J.L., Serafini, T., Kennedy, T.E., Tessier-Lavigne, M., Goodman, C.
S., Dickson, B.J., 1996. Genetic analysis of Netrin genes in Drosophila: Netrins
guide CNS commissural axons and peripheral motor axons. Neuron 17,
Molina, M.R., Cripps, R.M., 2001. Ostia, the inflow tracts of the Drosophila heart,
develop from a genetically distinct subset of cardial cells. Mech. Dev. 109,
Moore, S.W., Tessier-Lavigne, M., Kennedy, T.E., 2007. Netrins and their receptors.
Adv. Exp. Med. Biol. 621, 17–31.
Purohit, A.A., Li, W., Qu, C., Dwyer, T., Shao, Q., Guan, K.L., Liu, G., 2012. Down
Syndrome Cell Adhesion Molecule (DSCAM) associates with uncoordinated-5C
(UNC5C) in Netrin-1-mediated growth cone collapse. J. Biol. Chem. 287,
Quinn, C.C., Wadsworth, W.G., 2008. Axon guidance: asymmetric signaling orients
polarized outgrowth. Trends Cell Biol. 18, 597–603.
Santiago-Martinez, E., Soplop, N.H., Patel, R., Kramer, S.G., 2008. Repulsion by Slit
and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during
Drosophila heart tube lumen formation. J. Cell Biol. 182, 241–248.
Soplop, N.H., Patel, R., Kramer, S.G., 2009. Preparation of embryos for electron
microscopy of the Drosophila embryonic heart tube. J. Vis. Exp..
Timofeev, K., Joly, W., Hadjieconomou, D., Salecker, I., 2012. Localized netrins act as
positional cues to control layer-specific targeting of photoreceptor axons in
Drosophila. Neuron 75, 80–93.
Vanderploeg, J., Vazquez Paz, L.L., MacMullin, A., Jacobs, J.R., 2012. Integrins are
required for cardioblast polarisation in Drosophila. BMC. Dev. Biol. 12, 8.
Watari-Goshima, N., Ogura, K., Wolf, F.W., Goshima, Y., Garriga, G., 2007. C. elegans
VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell and growth-cone
migrations. Nat. Neurosci. 10, 169–176.
Woods, D.F., Hough, C., Peel, D., Callaini, G., Bryant, P.J., 1996. Dlg protein is required
for junction structure, cell polarity, and proliferation control in Drosophila
epithelia. J. Cell Biol. 134, 1469–1482.
Xu, Z., Li, H., Wadsworth, W.G., 2009. The roles of multiple UNC-40 (DCC) receptor-
mediated signals in determining neuronal asymmetry induced by the UNC-6
(netrin) ligand. Genetics 183, 941–949.
Zaffran, S., Astier, M., Gratecos, D., Semeriva, M., 1997. The held out wings (how)
Drosophila gene encodes a putative RNA-binding protein involved in the
control of muscular and cardiac activity. Development 124, 2087–2098.
Ziel, J.W., Hagedorn, E.J., Audhya, A., Sherwood, D.R., 2009. UNC-6 (netrin) orients
the invasive membrane of the anchor cell in C. elegans. Nat. Cell Biol. 11,
F.D. Macabenta et al. / Developmental Biology 380 (2013) 233–242