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First published online April 12, 2006
doi: 10.1242/10.1242/dev.02353
1 Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, PO
Box 85800, San Diego, CA 92186, USA.
2 Program in Neuroscience and Arizona Research Laboratories Division of
Neurobiology, The University of Arizona, Tucson, AZ 85721, USA.
* Author for correspondence (e-mail: jthomas{at}salk.edu)
Accepted 9 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Eph, Axon branching, Mushroom body
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). MB
neurons bifurcate and extend axons into two distinct areas of the brain,
ultimately synapsing with different targets and giving the MB its
characteristic bi-lobed appearance. The apparent synchrony of the growth of
the two branches suggests that the growth cones from individual branching MB
neurons may differentially respond to guidance cues
(Wang et al., 2002). A class of molecules known to play key roles in the organization of many tissues, including axon guidance in the nervous system, is the Eph receptor tyrosine kinase (RTK) family members and their ligands, the Ephrins. Members of these large, phylogenetically conserved families of receptors and ligands are found throughout the animal kingdom, but the families have undergone a considerable expansion within vertebrates, resulting in a total of 14-16 Eph receptors and eight to nine Ephrin ligands depending upon the species. Eph receptors are divided into two subclasses based on sequence and their preferential binding affinity for Ephrin ligands: EphA receptors bind to GPI linked Ephrin A ligands, while EphB receptors bind to transmembrane Ephrin B ligands. Ephs and Ephrins are expressed in many developing tissues and mediate a variety of cell-cell contact-dependent signaling events, including axon attraction and repulsion within the nervous system.
The diverse roles played by Ephs and Ephrins are reflected in the
complexity of their signaling. Both the receptor and ligand are membrane bound
molecules capable of transducing intracellular signals within a cell. Thus,
signaling through both the receptor (forward) and the ligand (reverse) are
possible. In some cases, such as rhombomere boundary formation within the
hindbrain, activation of Eph/Ephrin signaling occurs when Eph-expressing cells
encounter domains of Ephrin expression
(Poliakov et al., 2004).
However, in many cases Eph/Ephrin signaling occurs within cells expressing
both receptor and ligand. The consequence of such ligand/receptor
co-expression appears to be context dependent. For example, within the
retinotectal system Eph/Ephrin co-expression by retinal ganglion cells may
result in the Ephrin ligand masking the Eph receptor, blocking receptor
activation by ligand expressed in trans
(Hornberger et al., 1999). By
contrast, within developing motoneurons, co-expressed ligand and receptor do
not appear to interact, thereby allowing both receptor and ligand in principal
to signal independently within the same cell
(Marquardt et al., 2005).
Consistent with this idea, within such co-expressing motoneurons the Eph
receptor mediates growth cone collapse and repulsion, while the Ephrin
mediates axon growth and attraction
(Marquardt et al., 2005).
Eph/Ephrin signaling in axon guidance is perhaps best illustrated by its
role in the topographic mapping of retinal ganglion cells in the superior
colliculus (SC) (Poliakov et al.,
2004). Retinal ganglion cell axons initially overshoot their
targets within the superior colliculus and subsequently contact their correct
termination zones through the extension of collateral branches. Along the
anteroposterior (AP) axis of the SC, retinal ganglion cell targeting relies in
part on EphA/Ephrin A signaling mediating repulsion
(Brown et al., 2000;
Nakamoto et al., 1996;
Yates et al., 2001). By
contrast, along the mediolateral axis of the SC, opposing gradients of EphB
receptors on retinal ganglion cells and Ephrin B ligands on SC cells act to
control the direction of branch extension and arborization by mediating either
attraction or repulsion, depending upon the position of the branch relative to
its termination zone (Hindges et al.,
2002; Mann et al.,
2002; McLaughlin et al.,
2003).
In contrast to animals that have multiple Eph receptors and/or ligands,
Drosophila has a single Eph and a single Ephrin. The
Drosophila Eph receptor shows equal similarity to both the A and B
subclasses (Dearborn, Jr et al.,
2002; Scully et al.,
1999), while the Drosophila Ephrin ligand is most similar
to vertebrate Ephrin B ligands. Like other Ephrin B ligands,
Drosophila Ephrin contains a transmembrane domain and a conserved
tyrosine phosphorylation site (Bossing and
Brand, 2002).
Both Drosophila Eph and Ephrin are expressed within the
embryonic CNS at a time when neurons are extending axons towards their targets
(Bossing and Brand, 2002;
Scully et al., 1999). Two
previous studies have suggested a role for Drosophila Eph/Ephrin
signaling in neuronal development using RNA interference (RNAi) technology
(Bossing and Brand, 2002;
Dearborn et al., 2002). Here,
we describe the generation of a null mutation in Eph, plus our
analysis of Eph/Ephrin function within the Drosophila CNS in
individuals lacking all Eph function. We show that Drosophila Eph and
Ephrin can act as a functional receptor ligand pair in vivo to mediate axon
repulsion. Despite this, we fail to detect axon guidance defects in the
embryonic CNS of Eph mutant embryos. However, later in development
Eph/Ephrin signaling plays a crucial role in the developing MB by guiding the
projection of specific axon branches of individual MB neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is a lethal white+ (w+) P element
insertion on chromosome IV that was mapped to the bent locus by
sequencing fragments generated by inverse PCR
(Dalby et al., 1995). 39c-18
was mobilized using 
2,3
(Robertson et al., 1988).
Six-hundred
w;CyO,2,3/+;39c-18/CiD
males were tested for chromosomes with reversion of 39c-18 lethality by singly
crossing them to w-females carrying a lethal allele of bent
(39c-18LR#56/CiD) generated by excision of 39c-18.
Five-hundred and eighty w+ non-CyO,2,3 flies
were isolated, 57 of which mapped to chromosome IV. Insertion sites for 54
lines were determined by inverse PCR; flanking sequences from three lines did
not map to a single site and were discarded.
P114 excisions were generated by crossing w;CyO,
2,3/+;P114/CiD males to
w;eyD/CiD females. One-thousand three-hundred
w-excision lines were isolated, 2.5% of which were homozygous lethal.
Southern blot analysis demonstrated that none of the lethal lines deleted
sequences within the Eph genomic region. Seven out of 16 lethal lines
did show rearrangements within the onecut genomic region, including
the onecutx122 and onecutx49 alleles.
Eight additional onecut alleles were identified by
non-complementation with onecutx122 and
onecutx49.
Viable lines were screened for rearrangements within Eph by PCR
using primers that amplify the first three exons of the Eph gene
(Scully et al., 1999). After
additional southern blot analysis of Ephx652 DNA indicated
a deletion of the first three exons, breakpoints were determined by sequencing
a PCR fragment generated from Ephx652 DNA using primers
predicted to bracket the excised region. The 5' breakpoint of
Ephx652 maps to genomic position 627320 (BDGP release 4),
368 bp upstream of the onecut translation start site. The 3'
breakpoint lies within the third intron of Eph, at genomic position
632468.
Immunohistochemistry and in situ hybridization
Immunohistochemistry on dissected embryos was preformed as previously
described (Callahan and Thomas,
1994). Larval and adult brains were dissected and fixed as
described by Hummel et al. (Hummel et al.,
2003) with a total fixation time was 60 minutes.
Primary antibodies used: monoclonal antibody (mAb) anti-BP102 (1:20
dilution) (Seeger et al.,
1993); mAb anti-Fas2 (1:30)
(Lin et al., 1994); mAb 9E10
anti-c-myc (Dm - FlyBase) (1:50) (Developmental Studies Hybridoma Bank);
Cy3-conjugated anti-HRP (1:500) (Jackson Immunoresearch); rabbit
anti-ß-Gal antibody (1:1000) (Cappel); and rabbit anti-GFP (1:5000)
(Molecular Probes).
Ephrin-Fc was produced as described
(Kaneko and Nighorn, 2003).
Samples were incubated in either purified Ephrin-Fc (1:500 dilution in PBS) or
straight supernatant, followed by incubation with either rabbit or mAb
anti-human IgG, Fc
(1:500) (Jackson ImmunoResearch). For fluorescence
immunostaining, Alexa Fluor 488-conjugated anti-rabbit (1:500) (Molecular
Probes) and Cy3-conjugated anti-mouse (1:500) (Jackson ImmunoResearch) were
used. In situ hybridization was carried out as described
(Dougan and DiNardo, 1992)
using probes generated with fragments corresponding to 
1 kb of 5'
sequences of the Eph cDNA (Scully
et al., 1999) or the Ephrin cDNA.
Constructs
pUAS-Ephrin:myc was constructed from the full-length cDNA clone
RE46807 tagged in-frame to six copies of the c-myc epitope in the
pUAS vector (Brand and Perrimon,
1993). For construction of pUAS-dephrinmycIC, a
PCR fragment was generated that deleted intracellular sequences from amino
acids 611 to 652. This was subcloned as an EcoR1/AgeI
fragment into pUAS-Ephrin:myc, replacing the wild-type sequence. For
each UAS transgene, multiple lines were generated by P element
transformation (Spradling and Rubin,
1982). Lines with the strongest anti-myc staining were used.
Genetic mosaics
The following flies were generated for MARCM analysis of MB neurons:
hsp70-FLP,elav-Gal4,UAS-mCD8GFP/+ or
Y;FRTG13/FRTG13,TubP-Gal80;Ephx652/CiD
and hsp70-FLP,elav-Gal4,UAS-mCD8GFP/+ or
Y;FRTG13/FRTG13,TubP-Gal80;Ephx652/Ephx652.
Clones within the 
/ß lobes were generated by heat shocking pupae
at 38°C, as described (Lee and Luo,
2001). Eighty adult Ephx652 individuals were
examined for marked MB clones. Branching patterns of 41 unambiguously labeled
Ephx652 mutant MBs clones were examined.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), located 145 kb away from Eph and inserted in
the bent locus (see Materials and methods). Fifty-five independent
4th chromosome insertion lines were generated. Insertion sites for all 55 were
determined by inverse PCR and represent a unique collection of 4th chromosome
P-element insertion lines which will be made available from the Bloomington
Drosophila Stock Center. One line, P114, maps within 3 kb of the
Eph transcription start site and 1 kb upstream of
onecut, a gene transcribed on the opposite strand encoding a
homeodomain class transcription factor
(Nguyen et al., 2000). As both
Eph and onecut expression are unaffected in homozygous P114
flies (data not shown), we used this line to generate Eph deletions
by imprecise excision. A total of 1300 excision lines were generated, 33 of
which, or 2.5%, were found to be homozygous lethal. Both viable and lethal
excision lines were assayed by PCR and southern blot analysis for
rearrangements within the Eph genomic region. A single viable
excision line, Ephx652, was isolated that removes
Eph genomic sequences (Fig.
1A). This excision deletes the first three exons of Eph,
thus removing the Eph transcriptional start site and the first 79
amino acids of the coding region. The absence of detectable Eph mRNA
and protein expression in Ephx652 homozygous individuals
argues that it acts as a null mutation
(Fig. 1C; see below). Sequencing across the breakpoint of Ephx652 revealed that this excision also removes 5' sequences of the onecut locus, including its transcription start site. It does not, however, remove any onecut coding sequence. Among the excision lines, we identified 15 that extend in the direction of onecut, removing onecut-coding sequence but not removing sequence in the direction of or within the Eph gene (Fig. 1A). All of these excision lines are lethal as homozygotes. In addition, intra-allelic combinations of the excisions, such as onecutlx122/onecutlx49, are lethal, demonstrating an essential function for the onecut locus. In contrast to the lethality of the onecutlx122 and onecutlx49 alleles, homozygous Ephx652 flies are viable and fertile, and have no obvious morphological defects, suggesting that any effect of Ephx652 on onecut function must be partial. Consistent with this, Ephx652 complements the lethality of the onecut alleles, as Ephx652/onecutlx122 and Ephx652/onecutlx49 flies are viable and fertile.
The single Drosophila Ephrin gene contains a P element insertion,
KG09118, generated by the Berkeley Gene Disruption Project, that lies 20
bp downstream of the Ephrin transcription start site
(Bellen et al., 2004;
Roseman et al., 1995). In situ
hybridization of Ephrin antisense RNA probes to homozygous
EphrinKG09118 embryos reveals that this insertion severely
reduces Ephrin mRNA expression
(Fig. 1F), indicating that this
insertion is an Ephrin allele. Like Ephx652,
homozygous EphrinKG09118 individuals are viable and
fertile.
Drosophila Eph is expressed on embryonic CNS axons during pathfinding
Previous in situ hybridization studies showed that Eph is
expressed within the CNS at a time when neurons extend axons towards their
targets and is expressed by most, if not all, CNS neurons at this stage
(Scully et al., 1999). To
visualize the distribution of Drosophila Eph receptor, we used
Ephrin-Fc, a soluble probe consisting of the extracellular domain of
Manduca Ephrin fused to the human immunoglobulin Fc fragment
(Kaneko and Nighorn, 2003).
The major axon tracts within the CNS, the bilaterally symmetric longitudinal
connectives and the two commissures connecting each hemisegment, can be
visualized with the panaxonal BP102 antibody
(Seeger et al., 1993).
Incubation of unfixed, dissected embryos with BP102 and Ephrin-Fc reveals that
the Eph receptor is expressed on both longitudinal and commissural axons
within the CNS in a pattern similar to that of BP102 staining
(Fig. 2A-C). Thus, the Eph
receptor is expressed by most, if not all embryonic CNS neurons and is
targeted to axons.
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Ephrin mRNA, like Eph, is widely expressed throughout the
embryonic CNS, as assayed by in situ hybridization
(Fig. 1F) and Ephrin protein is
expressed by most if not all CNS neurons, but in contrast to Eph has been
reported to be localized to cell bodies rather than axons
(Bossing and Brand, 2002).
Consistent with this, we have found that when expressed by neurons using the
GAL4/UAS transactivation system (Brand and
Perrimon, 1993), a myc-tagged version of Ephrin is localized to
cell bodies (Fig. 2G-I).
Therefore, both Drosophila Eph and Ephrin are present at a time and
place that would allow them to control axon pathfinding, but each appears to
reside within a distinct compartment of the neuron.
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).
These defects are consistent with Ephrin acting as an axonal repellent,
preventing axons from crossing the ectopic source of Ephrin at the midline.
Similar axon guidance defects are observed when an Ephrin transgene lacking
all intracellular sequences,
UAS-EphrinIC:myc, is expressed
by sim-Gal4 (Fig. 3C).
This result, combined with the finding that misexpression of Eph by midline
glia has no effect on commissural axons (data not shown), confirms that it is
forward signaling that causes axons to be repelled and that reverse signaling
plays no role in this repulsion. To test whether the repellent activity of
Ephrin is mediated by Eph, we misexpressed Ephrin in midline glia using the
same combination of transgenes as above, but in an Ephx652
mutant background. In these embryos, the ability of Ephrin to repel
commissural axons is abolished, restoring the commissural tracts to wild type
in their appearance (Fig. 3D).
Thus, Eph and Ephrin are able act as a receptor/ligand pair in vivo and, at
least in this assay, Ephrin acts solely through the Eph receptor.
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). As
homozygous Ephx652 flies are viable and fertile, we took
embryo collections from a homozygous mutant stock, thus eliminating both
zygotic and maternal Eph. Homozygous Ephx652 mutant
embryos show no obvious defects in overall axon tract organization compared
with wild-type embryos (Fig.
4A,B).
We next assayed axon pathfinding in greater detail using markers that label
the axonal projections of subsets of neurons. First, we stained embryos for
Fasciclin 2 (Fas2), a cell-adhesion molecule expressed on five distinct
bilaterally symmetric bundles of axons that run along the anteroposterior axis
of the CNS (Lin et al., 1994).
We detected no obvious difference in the Fas2 axon bundles between wild-type
and Ephx652 mutant embryos, although the bundles in
Ephx652 mutants appear slightly less fasciculated
(Fig. 4C,D). We next examined
the axon trajectories of the Apterous (Ap) neurons, three neurons per
abdominal hemisegment that extend axons anteriorly as a tightly fasciculated
bundle along the most medial Fas2 bundle
(Lundgren et al., 1995;
Simpson et al., 2000). The
axonal projections of the Ap neurons in Ephx652 mutant
embryos are indistinguishable from wild type
(Fig. 4E,F). In addition to the
Ap neurons, we examined the trajectories of other subsets of neurons such as
the Eagle neurons (Bonkowsky et al.,
1999), the VUM neurons
(Callahan and Thomas, 1994) and
the MP1 neurons (Hidalgo and Brand,
1997). In all cases, we could detect no axon guidance defects in
Ephx652 mutant embryos (data not shown).
We conclude from these results that although activation of Eph signaling by ectopic Ephrin expression results in axon repulsion, Eph signaling plays only a limited role, if any, in embryonic axon guidance. Consistent with this, we found no obvious axon pathfinding defects in EphrinKG09118 mutant embryos (data not shown).
Eph is expressed by mushroom body neurons throughout development
Studies in vertebrates have implicated a role for Eph/Ephrin signaling in
the development of the olfactory system. In Drosophila, olfactory
neurons within the antennae and maxillary palp project to glomeruli within the
antennal lobe. Projection neurons, which form dendritic connections with
antennal glomeruli, in turn project their axons to the dendritic region of the
bilaterally symmetric mushroom bodies (MB) where olfactory information is
processed (Crittenden et al.,
1998; Heisenberg,
2003; Jefferis et al.,
2002).
The neurons giving rise to the distinct lobes of the MB are clonally
related (Ito et al., 1997;
Lee et al., 1999). Four
neuroblasts set aside early in development generate a pool of MB neurons in
three distinct mitotic waves, giving rise in sequence to the larval born
neurons, the early pupal '/ß' neurons and
finally the later born /ß neurons
(Fig. 5A). The initial axonal
projections of neurons prefigure those of the later born
'/ß' and /ß neurons. They send axons
anteriorly as a tightly packed bundle within a structure known as the
peduncle. Axons then bifurcate forming a dorsal and medial branch seen in late
embryo/early 1st instar through 3rd instar larval stages. These axonal
processes undergo degeneration during metamorphosis, only to re-extend a
single axonal projection medially during puparation to give rise to the adult
structure (Jefferis et al.,
2002).
The second group of neurons, the '/ß' neurons are
generated during late 3rd instar. Like neurons,
'/ß' neurons extend an axon through the peduncle, and
bifurcate, sending a distinct projection medially, along with a second
collateral extension dorsally, generating the ' and ß'
lobes. Finally, similar projections by the third group of MB neurons, the
/ß neurons, result in the formation of an additional, distinct
dorsal and medial projection, forming the and ß lobes. The
and ß lobes are further characterized by strong expression of
Fas2 (Crittenden et al., 1998;
Noveen et al., 2000).
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neurons already display their characteristic bifurcated appearance,
we detect Eph expression by most neurons of the brain, including the
developing MB neurons (Fig.
5B-D). As in the embryonic CNS, Eph is targeted to axons. By 3rd
instar larval stages, Eph staining within the brain becomes restricted to the
bifurcated neurons (Fig.
5E-G). At early pupal stages, levels of Eph expression within the
MB increase and the receptor is present on '/ß' axons
(Fig. 5H-J). Eph receptor is
present throughout the MB peduncle, as well as on both dorsal and medial lobe
projections at pupal stages. Expression persists as development proceeds, but
becomes limited primarily to the /ß neurons at late pupal stages
(Fig. 5K-M) and the adult
(Fig. 5N-P), as revealed by the
colocalization with Fas2. Interestingly, in the adult MB, Eph levels are
higher on the dorsal projecting lobes, particularly within their
terminal regions (Fig. 5O,
arrow). Thus, Eph receptor is restricted to specific lobes within the MB in
the adult and its localization within a single /ß neuron is
tightly regulated.
We have examined Ephrin expression by in situ hybridization on
larval brains at a time when MB neurons bifurcate, forming dorsal and medial
lobes. Ephrin expression within the brain at this time is detectable
in many subsets of neurons including MB neurons
(Fig. 5S, arrows). These
results demonstrate that MB neurons, like embryonic neurons, co-express both
ligand and receptor during MB development. Furthermore, as in embryonic
neurons, Ephrin:myc is localized to MB cell bodies when expressed by
OK107-Gal4 (Fig.
5Q,R), a Gal4 driver expressed specifically by the MB throughout
its development (Connolly et al.,
1996; Kurusu et al.,
2002). Therefore, both Drosophila Eph and Ephrin are
present within developing MB neurons, but each appears to reside within
distinct neuronal compartments.
Eph/Ephrin function is required for dorsal lobe formation within the MB
To examine the overall structure of the adult MB in both wild type and
Eph mutants, we used elav-Gal4 plus UAS-mCD8:GFP.
This combination of transgenes labels all five lobes of the adult MB
(Fig. 6A,B); the and
ß lobes can be specifically visualized by double labeling with Fas2. We
found that Ephx652 adult flies have severe MB defects
(n=50). The most dramatic phenotype is reduced or absent
' and lobes, often with an associated increase in the
thickness of ß' and ß lobes
(Fig. 6B,K). This phenotype is
present in 60% of adult flies, and primarily exhibits itself unilaterally
within the MB. Rarely do we detect severe bilateral defects, although in these
cases similar reduced or absent dorsal lobe projections are observed. The
severity of MB lobe projection defects covers a continuous range, from
lobes completely missing to lobes that appear normal. Defects in lobe
projections are always associated with defects in the earlier forming
' lobe, suggesting that '/ß' neurons may
act as pioneers for the later born /ß projections as has been
proposed (Wang et al., 2002).
MB development in Ephx652/onecutx122
and Ephx652/onecutx49 heterozygotes
(n=26) is indistinguishable from wild type
(Fig. 6I), indicating that the
phenotypes we observe within the MB of Ephx652 mutants are
specifically due to loss of Eph.
Defects in dorsal lobe development of Eph mutants are evident at
all stages of MB development. Defects are detected in developing
neuron dorsal projections in early pupae (6C,D), as well as in dorsal
projecting branches of the earlier born neurons
(Fig. 6E-H). By the 3rd instar
larval stage, wild-type neurons have acquired a bifurcated appearance,
generating a dorsal and medial projection
(Fig. 6E). In 60% of
Ephx652 mutant 3rd instar larvae, we see either reduced or
absent dorsal lobe projections (Fig.
6F). Many of these same MBs show obviously thicker medial lobes in
association with dorsal lobe defects (Fig.
6F, arrowhead). Similar defects in dorsal lobe projections are
apparent within developing late embryonic/early 1st instar larval MBs, at a
time when neurons are first pathfinding (6G-H). Thus, Eph function is
required at all stages of MB development.
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lobe projections identical in nature to those of
Ephx652 individuals
(Fig. 6J). Taken together,
these results indicate a requirement for Eph/Ephrin signaling in the
development of dorsal lobe projections within the MB.
Targeted overexpression of Eph disrupts MB development
To confirm that lack of Eph expression and function in MBs results in the
dorsal lobe defects, we attempted to rescue the MB defects of
Ephx652 homozygous adult flies by replacing Eph expression
using the Gal4/UAS system. For these experiments, we used elav-Gal4,
which is expressed in all postmitotic neurons throughout development, and the
MB-specific OK107-Gal4 driver. Expression of Eph within the MB using
either of these drivers fails to rescue the /' lobe
defects. In fact, expression of Eph within the MB of either wild type or
Ephx652 mutants results in a phenotype similar to the
Eph loss of function phenotype. In these individuals, there is a high
frequency and often bilateral loss of lobes and a concomitant increase
in ß lobe thickness (Fig.
6K). In 50% of these MBs, ß lobes appear fused at the
midline (arrowhead), signifying that medially projecting branches now fail to
respect the midline boundary. Such failure to respect the midline boundary is
also observed in 10% of Ephx652 mutant MBs (arrowhead in
Fig. 6L). These results argue
that MB neurons are sensitive to levels or timing of Eph expression.
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We visualized individual neurons and their projections using the MARCM
system (Lee and Luo, 1999), in
which clones of cells are generated by mitotic recombination and identified by
expression of UAS-mCD8:GFP. We randomly generated marked clones in
wild type and in homozygous Ephx652 mutants at late pupal
stages, thus labeling the trajectories of /ß neurons. Ideally, we
would have generated Ephx652 mutant neurons in an
otherwise phenotypically wild-type animal, thus determining the autonomous
function of Eph within MB neurons. However, owing to the difficulties in
generating clones by recombination of the 4th chromosome, we were limited to
examining the fate of individual MB neurons within homozygous
Ephx652 mutant individuals.
In wild type, individual /ß neurons extend a single axonal
projection into the peduncle that eventually bifurcates, one branch extending
medially in the ß lobe, and the other dorsally in the lobe
(Fig. 7A,B). Little or no
additional branching is observed along the length of each projection, although
limited arborization at the end of each lobe can occur. Individual
/ß neurons in Ephx652 adults project a single
axon along the length of the peduncle as in wild type
(Fig. 7C, inset) and at the
base of the peduncle mutant neurons bifurcate normally. However, instead of
projecting one branch dorsally along the lobe, both branches extend
medially along distinct paths within the ß lobe
(Fig. 7C,D). In addition,
supernumerary branches along the lengths of the ß lobe projections are
present in Ephx652 mutants (arrowheads in
Fig. 7D). Thus, Eph/Ephrin
signaling is not required for branch formation, but for correct pathfinding of
dorsal lobe branches within the developing MB.
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lobe is present but
reduced, marked MB neurons either project normally or show lobe
projection defects (n=8). In larger clones within such MBs, we
observe more labeled neurons projecting axons within the ß lobe than the
lobe (Fig. 7E,F),
indicating that some neurons within a single Ephx652
mutant MB can extend branches normally within dorsal and medial lobes, while
other neurons do not, instead extending both branches within the medial ß
lobe. These results argue that individual branches of MB neurons make
independent pathfinding decisions. Furthermore, these results are consistent
with a continual requirement of Eph signaling within MB neurons for the
correct guidance of individual branches.
In Ephx652 mutants in which the overall MB structure
appears normal, individually marked neurons within all clones examined extend
branches normally in and ß lobes
(Fig. 7G,H; n=18). In
these MBs, individually marked neurons also have a normal unbranched
appearance, suggesting that the ectopic branching observed in phenotypically
mutant MBs may be a consequence of incorrect pathfinding and target selection
of the dorsal branch. It is possible that within these overall
normal-looking MBs there are neurons with abnormal projections, but their
numbers are sufficiently small that the probability of one being included in a
marked clone is very low.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Palmer and Klein, 2003;
Poliakov et al., 2004). Given
the simplicity of Drosophila Eph/Ephrin, with its single Eph receptor
and single Ephrin ligand, each sharing similarities to both A and B vertebrate
subclasses, one might have expected the role of this prototypical Eph/Ephrin
complex to reflect the most basic axon guidance functions of this family of
proteins. Surprisingly, our analysis of Eph mutants shows that Eph/Ephrin
signaling has a very specific requirement in MB development rather than a
general role in axon guidance. Although the receptor/ligand pair is expressed
in the embryonic CNS and Eph is capable of signaling when ectopically
activated, this signaling appears to have a limited role in the guidance of
axons. Similarly, although Drosophila Eph is expressed within the
developing visual system, and RNAi knockdown experiments previously suggested
a role in retinotopic map formation
(Dearborn et al., 2002), we
detect no defects in the overall targeting of photoreceptor cells in the
lamina and medulla in the absence of Eph function (not shown). Thus,
in these developing systems, Eph/Ephrin signaling either acts with other
guidance molecules or functions in processes other than axon guidance.
Genetic loss of function versus RNAi
Our analysis of the embryonic CNS in Eph-null mutants shows that Eph
signaling plays little or no role in axon guidance. Although we have not
examined all guidance events, and in fact subtle guidance and targeting
defects may yet be uncovered, the lack of any obvious defects is in marked
contrast to a previous study using RNAi to knock down Eph expression
(Bossing and Brand, 2002). RNAi
is a widely used method to suppress gene expression in many organisms,
including Drosophila. Although it is generally accepted that RNAi
acts specifically to downregulate target gene expression because of its
requirement for near perfect sequence identity with the target transcript,
there is evidence that interference can occur with sequences carrying only
limited sequence similarity (Jackson et
al., 2003). The Eph receptor contains regions of sequence
similarity, such as the fibronectin type III repeats, to other proteins
expressed within the nervous system
(Scully et al., 1999). Perhaps
the disparity between our results and the results using RNAi is due to
expression changes in genes other than Eph that contain sequences
capable of binding the Eph RNAi.
Eph signaling in Drosophila MB axon guidance
Our analysis of Eph mutants reveals a specific role for Eph
signaling in regulating the guidance of individual axon branches within the
MB. As in wild type, growing axons of MB neurons in Eph mutants
bifurcate at the base of the peduncle, forming two branches. However, these
two branches often fail to choose divergent paths and instead both extend
medially. This is the first evidence for receptor signaling in the guidance of
individual branches of MB neurons. Other receptors such as the Down syndrome
cell-adhesion molecule (Dscam) and signaling molecules such as the Rho family
of GTPases appear to have broader roles in regulating MB axon fasciculation,
branch formation, extension and segregation
(Schmucker et al., 2000;
Wang et al., 2002;
Zhan et al., 2004). For
example, Dscam is required for MB neuron branch segregation
(Wang et al., 2002;
Zhan et al., 2004) through a
mechanism thought to involve the mutual repulsion of sister branches
(Wang et al., 2002;
Wojtowicz et al., 2004). In
the absence of Dscam, when branches form, they extend randomly in either
dorsal or medial directions and can be fasciculated. By contrast, Eph has a
more specific role in the guidance of dorsal projections and sister branches
do not tightly associate with each other even when both project medially.
Therefore, the mechanisms that ensure the formation and maintenance of
distinct axon branches are unaffected in Eph mutants and we would
predict that Dscam functions normally in Eph mutant MB neurons.
How branch formation occurs in the Drosophila MB is unclear, as
branching MB neurons have yet to be followed in situ and in real time.
Individual MB growth cones might respond to specific cues by splitting,
resulting in formation of two growth cones that project independently, a
possibility supported by the relative synchrony of branch formation
(Wang et al., 2002).
Alternatively, branches might arise as collateral extensions off existing
axons (Noveen et al., 2000).
Regardless of the precise mechanism of branching within the MB, sister
branches must extend away from each other, one in the dorsal lobe and one in
the medial lobe. Although Eph is clearly required for the proper guidance of
dorsal branches, the mechanism by which it acts is unknown. In one model, Eph
receptor signaling, in response to a localized Ephrin source, acts as an
attractive signal to guide the extension of one sister branch in a dorsal
direction. In the absence of this attractive cue, both branches extend in the
`default', or medial, direction. Alternatively, Eph/Ephrin signaling may act
as a repellent guidance signal, in which case Eph-mediated repulsion could
steer the future dorsal branch away from the midline, allowing its extension
dorsally. If branching does occur by growth cone splitting, both of these
models call for the differential localization or activation of the Eph
receptor within a single branch, and that this spatially localized signaling
leads to the guidance of this branch dorsally. In this regard, we have not
detected any clear differences in Eph receptor distribution between dorsal and
medial lobes at a time when MB neurons are in the process of projecting axons.
However, like branch initiation itself, differential Eph expression could be a
transient event and difficult to visualize by examining the entire collection
of bifurcating MB neurons.
Co-expression of Eph and Ephrin
At present, the precise localization of the Ephrin ligand during branch
formation is not known. Our studies of Ephrin RNA expression,
however, indicate that, as in the embryonic CNS, MB neurons co-express both
receptor and ligand. Therefore, the means by which Eph/Ephrin signaling
regulates branch extension dorsally may be more complicated than
Eph-expressing MB neurons simply responding to Ephrin in their environment.
There are now a growing number of instances where Eph receptors and their
corresponding ligands are co-expressed within a given tissue
(Knoll and Drescher, 2002;
Marquardt et al., 2005). This
co-expression allows, at least in theory, the possibility for both trans and
cis regulation of the Eph/Ephrin signaling complex, as well as for both
forward and reverse signaling in the same cell
(Santiago and Erickson, 2002).
The ultimate path that a growing axon takes therefore, may depend on the
balance between these various signals.
Eph/Ephrin signaling in axon branch guidance
The requirement for Eph and Ephrin in guiding branches within the
developing Drosophila MB is reminiscent of the role of Eph/Ephrin
signaling in the retinotopic mapping of retinal ganglion cells within the
midbrain of vertebrates (Hindges et al.,
2002; McLaughlin et al.,
2003; Poliakov et al.,
2004) and layer-specific branching of thalamic axons within the
cortex (Mann et al., 2002). In
the retinotectal system, initial imprecise axon extensions of retinal ganglion
cells into the midbrain are refined by extension of specific collateral
branches that resolve into synaptic arbors along appropriate retinotopic
locations along both the anteroposterior and dorsoventral axes. EphA/Ephrin A
signaling appears to control, in part, the point where branches form along the
AP axis of the midbrain, while graded EphB/Ephrin B cues guide branch
extension to appropriate locations along the DV axis. Here, as in the MB of
Drosophila, Eph/Ephrin signaling controls not the process of
branching itself, but the guidance of branches to appropriate target areas,
suggesting a conserved function for Eph/Ephrin in guiding branch extensions.
Drosophila MB neurons, with their stereotyped branch formation and
extension may provide a system for understanding the mechanisms whereby
Eph/Ephrin signaling activates intracellular signaling cascades that
ultimately lead to the guidance of individual branches to appropriate
targets.
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ACKNOWLEDGMENTS |
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REFERENCES |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et
al. (2004). The BDGP gene disruption project: single
transposon insertions associated with 40% of Drosophila genes.
Genetics 167,761
-781.
Bonkowsky, J. L., Yoshikawa, S., O'Keefe, D. D., Scully, A. L. and Thomas, J. B. (1999). Axon routing across the midline controlled by the Drosophila Derailed receptor. Nature 402,540 -544.[CrossRef][Medline]
Bossing, T. and Brand, A. H. (2002). Dephrin, a transmembrane ephrin with a unique structure, prevents interneuronal axons from exiting the Drosophila embryonic CNS. Development 129,4205 -4218.
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.[Abstract]
Brown, A., Yates, P. A., Burrola, P., Ortuno, D., Vaidya, A., Jessell, T. M., Pfaff, S. L., O'Leary, D. D. and Lemke, G. (2000). Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 102,77 -88.[CrossRef][Medline]
Callahan, C. A. and Thomas, J. B. (1994).
Tau-beta-galactosidase, an axon-targeted fusion protein. Proc.
Natl. Acad. Sci. USA 91,5972
-5976.
Connolly, J. B., Roberts, I. J., Armstrong, J. D., Kaiser, K.,
Forte, M., Tully, T. and O'Kane, C. J. (1996). Associative
learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.
Science 274,2104
-2107.
Crittenden, J. R., Skoulakis, E. M., Han, K. A., Kalderon, D.
and Davis, R. L. (1998). Tripartite mushroom body
architecture revealed by antigenic markers. Learn.
Mem. 5,38
-51.
Dalby, B., Pereira, A. J. and Goldstein, L. S. (1995). An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster. Genetics 139,757 -766.[Abstract]
Dearborn, R., Jr, He, Q., Kunes, S. and Dai, Y.
(2002). Eph receptor tyrosine kinase-mediated formation of a
topographic map in the Drosophila visual system. J.
Neurosci. 22,1338
-1349.
Dougan, S. and DiNardo, S. (1992). Drosophila wingless generates cell type diversity among engrailed expressing cells. Nature 360,347 -350.[CrossRef][Medline]
Heisenberg, M. (2003). Mushroom body memoir: from maps to models. Nat. Rev. Neurosci. 4, 266-275.[CrossRef][Medline]
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 124,3253 -3262.[Abstract]
Hindges, R., McLaughlin, T., Genoud, N., Henkemeyer, M. and O'Leary, D. D. (2002). EphB forward signaling controls directional branch extension and arborization required for dorsal-ventral retinotopic mapping. Neuron 35,475 -487.[CrossRef][Medline]
Hornberger, M. R., Dutting, D., Ciossek, T., Yamada, T., Handwerker, C., Lang, S., Weth, F., Huf, J., Wessel, R., Logan, C. et al. (1999). Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Neuron 22,731 -742.[CrossRef][Medline]
Hummel, T., Vasconcelos, M. L., Clemens, J. C., Fishilevich, Y., Vosshall, L. B. and Zipursky, S. L. (2003). Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam. Neuron 37,221 -231.[CrossRef][Medline]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D. (1997). The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124,761 -771.[Abstract]
Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G. and Linsley, P. S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 6, 635-637.
Jefferis, G. S., Marin, E. C., Watts, R. J. and Luo, L. (2002). Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies. Curr. Opin. Neurobiol. 12,80 -86.[CrossRef][Medline]
Kaneko, M. and Nighorn, A. (2003). Interaxonal
Eph-ephrin signaling may mediate sorting of olfactory sensory axons in Manduca
sexta. J. Neurosci. 23,11523
-11538.
Klein, R. (2004). Eph/ephrin signaling in morphogenesis, neural development and plasticity. Curr. Opin. Cell Biol. 16,580 -589.[CrossRef][Medline]
Knoll, B. and Drescher, U. (2002). Ephrin-As as receptors in topographic projections. Trends Neurosci. 25,145 -149.[CrossRef][Medline]
Kurusu, M., Awasaki, T., Masuda-Nakagawa, L. M., Kawauchi, H., Ito, K. and Furukubo-Tokunaga, K. (2002). Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II. Development 129,409 -419.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[CrossRef][Medline]
Lee, T. and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24,251 -254.[CrossRef][Medline]
Lee, T., Lee, A. and Luo, L. (1999). Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast. Development 126,4065 -4076.[Abstract]
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.[CrossRef][Medline]
Lundgren, S. E., Callahan, C. A., Thor, S. and Thomas, J. B. (1995). Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development 121,1769 -1773.[Abstract]
Mann, F., Ray, S., Harris, W. and Holt, C. (2002). Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-B ligands. Neuron 35,461 -473.[CrossRef][Medline]
Marquardt, T., Shirasaki, R., Ghosh, S., Andrews, S. E., Carter, N., Hunter, T. and Pfaff, S. L. (2005). Coexpressed EphA receptors and Ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell 121,127 -139.[CrossRef][Medline]
McLaughlin, T., Hindges, R., Yates, P. A. and O'Leary, D. D.
(2003). Bifunctional action of ephrin-B1 as a repellent and
attractant to control bidirectional branch extension in dorsal-ventral
retinotopic mapping. Development
130,2407
-2418.
Nakamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T., Hansen, M. J., Yoon, C. H., O'Leary, D. D. and Flanagan, J. G. (1996). Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo. Cell 86,755 -766.[CrossRef][Medline]
Nguyen, D. N., Rohrbaugh, M. and Lai, Z. (2000). The Drosophila homolog of Onecut homeodomain proteins is a neural-specific transcriptional activator with a potential role in regulating neural differentiation. Mech. Dev. 97, 57-72.[CrossRef][Medline]
Noveen, A., Daniel, A. and Hartenstein, V. (2000). Early development of the Drosophila mushroom body: the roles of eyeless and dachshund. Development 127,3475 -3488.[Abstract]
Palmer, A. and Klein, R. (2003). Multiple roles
of ephrins in morphogenesis, neuronal networking, and brain function.
Genes Dev. 17,1429
-1450.
Poliakov, A., Cotrina, M. and Wilkinson, D. G. (2004). Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev. Cell 7,465 -480.[CrossRef][Medline]
Robertson, H. M., Preston, C. R., Phillis, R. W.,
Johnson-Schlitz, D. M., Benz, W. K. and Engels, W. R. (1988).
A stable genomic source of P element transposase in Drosophila melanogaster.
Genetics 118,461
-470.
Roseman, R. R., Johnson, E. A., Rodesch, C. K., Bjerke, M., Nagoshi, R. N. and Geyer, P. K. (1995). A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster. Genetics 141,1061 -1074.[Abstract]
Santiago, A. and Erickson, C. A. (2002).
Ephrin-B ligands play a dual role in the control of neural crest cell
migration. Development
129,3621
-3632.
Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E. and Zipursky, S. L. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101,671 -684.[CrossRef][Medline]
Scully, A. L., McKeown, M. and Thomas, J. B. (1999). Isolation and characterization of Dek, a Drosophila eph receptor protein tyrosine kinase. Mol. Cell Neurosci. 13,337 -347.[CrossRef][Medline]
Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C. S. (1993). Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10,409 -426.[CrossRef][Medline]
Simpson, J. H., Kidd, T., Bland, K. S. and Goodman, C. S. (2000). Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance. Neuron 28,753 -766.[CrossRef][Medline]
Spradling, A. C. and Rubin, G. M. (1982).
Transposition of cloned P elements into Drosophila germ line chromosomes.
Science 218,341
-347.
Wallrath, L. L. and Elgin, S. C. (1995).
Position effect variegation in Drosophila is associated with an altered
chromatin structure. Genes Dev.
9,1263
-1277.
Wang, J., Zugates, C. T., Liang, I. H., Lee, C. H. and Lee, T. (2002). Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons. Neuron 33,559 -571.[CrossRef][Medline]
Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L. and Clemens, J. C. (2004). Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 118,619 -633.[CrossRef][Medline]
Yates, P. A., Roskies, A. L., McLaughlin, T. and O'Leary, D.
D. (2001). Topographic-specific axon branching controlled by
ephrin-As is the critical event in retinotectal map development. J.
Neurosci. 21,8548
-8563.
Zhan, X. L., Clemens, J. C., Neves, G., Hattori, D., Flanagan, J. J., Hummel, T., Vasconcelos, M. L., Chess, A. and Zipursky, S. L. (2004). Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron 43,673 -686.[CrossRef][Medline]
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