Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, C626, Urbana, IL 61801, USA
* Author for correspondence (e-mail: hing{at}life.uiuc.edu)
Accepted 19 December 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, Dock, Pak, Olfactory, Axon Pathfinding, Signaling, Antennal lobe, Glomeruli
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The exquisite connectivity of the odor map raises questions about its
development. What are the molecular mechanisms that allow olfactory axons to
connect precisely with their targets? A number of guidance molecules have been
found to be expressed in the developing olfactory system in vertebrates
(reviewed by Key and St John,
2002). Targeted disruptions of the neuropilin and ephrin A5 genes
in mouse show that they direct the projections of olfactory axons to broad
zones in the olfactory bulbs (Cloutier et
al., 2002
; Knoll et al.,
2001
; Schwarting et al.,
2000
). Furthermore, mutations of the odorant receptor genes reveal
that they control the convergence of olfactory axons on specific glomeruli
(Mombaerts et al., 1996
;
Wang et al., 1998
). Little is
known about the molecular mechanisms by which these proteins coordinately
guide olfactory axons to their correct glomerular targets
(Key and St John, 2002
).
Drosophila provides an excellent system with which to unravel odor
map development at the molecular and cellular levels. The anatomy and
development of the fly odor map not only bear close resemblance to those of
mammals, it is also simpler, containing only 60 Or genes
(Clyne et al., 1999
;
Gao and Chess, 1999
;
Scott et al., 2001
;
Vosshall et al., 1999
) and
40 uniquely identifiable glomeruli
(Laissue et al., 1999
). In
Drosophila, ONs differentiate in the antennal disc and send their
axons to the antennal lobe (AL), the fly equivalent of the mammalian olfactory
bulb (Jhaveri et al., 2000
;
Stocker et al., 1990
). Within
the AL, olfactory axons synapse on the dendrites of projection neurons (PNs)
in distinct glomeruli. As in mammals, each glomerulus is innervated by
olfactory axons expressing the same Or gene
(Gao et al., 2000
;
Scott et al., 2001
;
Vosshall et al., 2000
).
Remarkably, each glomerulus is also innervated by PNs born at a specific time
in development (Jefferis et al.,
2001
). Thus, in Drosophila, there is a precise pairing of
ONs expressing a given Or gene with PNs of a specific identity.
Previous studies have identified Dock and Pak (p21-activated Kinase) as
components of a signaling cascade that regulates the projection of
photoreceptor axons (Garrity et al.,
1996; Hing et al.,
1999
; Rao and Zipursky,
1998
). Dock is the fly homolog of the human Nck protein,
consisting entirely of three SH3 domains and a single C-terminal SH2 domain
(Garrity et al., 1996
). The
domain structure of Dock suggests that it acts as an adapter, linking
receptors to downstream signaling proteins
(Li et al., 2001
). Pak is a
serine/threonine kinase that is also highly conserved in evolution. Kinases of
this family are defined by the ability to bind, and be activated by Cdc42 and
Rac, key regulators of the actin cytoskeleton
(Bagrodia and Cerione, 1999
;
Daniels and Bokoch, 1999
).
Experiments, in both Drosophila and mammalian cells, show that Dock
and Pak function in a signaling cascade to regulate cell motility
(Hing et al., 1999
;
Sells et al., 1999
).
In this study, we show that Drosophila olfactory axons take stereotyped pathways in an outer nerve layer to find and innervate their cognate glomeruli. We found that dock and Pak are necessary for the precise wiring of the olfactory map. Using cell-type specific cDNA rescue, antibody localizations and clonal analyses, we observed that dock and Pak function autonomously in olfactory axons to control various choice points in their trajectories to the cognate glomeruli. Finally, structure/function studies show that Dock and Pak function in a signaling pathway in ON axon guidance. Thus, Dock and Pak may form a core signaling cascade employed by different ON subclasses to pathfind to their respective glomeruli.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clonal analysis
MARCM (Lee and Luo, 1999)
was carried out by heat-shocking third-instar larvae of the following
genotypes: hs-FLP/+; dock Or47a-Gal4/dock (or +); FRT82
Gal80/FRT82 UAS-mCD8::GFP and hs-FLP/+; dock Or47b-Gal4/dock (or
+); FRT82 Gal80/FRT82 UAS-mCD8::GFP at 37°C for 40 minutes. Adult
brains were dissected and processed as described below.
Immunohistochemistry
Adult brains (from 1- to 2-day-old animals), pupal antennae and larval
imaginal discs were dissected in phosphate buffered saline (PBS). Tissues were
fixed in PLP (2% paraformaldehyde, 0.25% sodium periodate, 75 mM lysine-HCl
and 37 mM sodium phosphate pH 7.4), washed with PBST (PBS with 0.5% Triton
X-100) and subjected to antibody staining. nc82 mAb (1:20) (A. Hofbauer, PhD
thesis, University of Wurzburg, 1991) was a gift from A. Hofbauer. Rabbit
anti-Dock 1:500 (Clemens et al.,
1996) was a gift from J. Dixon. Mouse 22C10 mAb (1:20) and rat
anti-ELAV, 7E8A10 (1:20) were from Developmental Studies Hybridoma Bank.
Rabbit anti-GFP polyclonal antibody (1:100) was from Clontech, and rat
anti-CD8
subunit mAb (1:100) was from Caltag. The secondary
antibodies, FITC-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse
and FITC-conjugated goat anti-rat, were purchased from Jackson Laboratories
and used at 1:200 dilutions.
Cuticle preparations
Adult antennae from animals expressing
Or-Gal4/UAS-lacZnuclear were fixed in 25% gluteraldehyde
for 1 hour, washed in PBST and stained in 0.2% X-Gal solution
(Ashburner, 1989). The antennae
were then cleared in Faure's mountant (34% v/v chloral hydrate, 13% glycerol,
20 mg/ml gum Arabic) and photographed with the SPOT-RT cooled CCD camera.
Sensilla on the third antennal segment were counted by projecting images on a
video monitor.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To ascertain the fate of the individual glomeruli, we expressed the
synaptobrevin::GFP fusion protein (encoded by the UAS-nsyb::GFP gene)
(Estes et al., 2000) under the
control of specific odorant receptor promoters (Or-Gal4 drivers)
(Vosshall et al., 2000
). The
presynaptic varicosities of the DM2, DM3 and VA11m glomeruli
(Laissue et al., 1999
), which
are located at various positions in the AL, were surveyed using the
Or22a-Gal4, Or47a-Gal4 and Or47b-Gal4 drivers, respectively.
In wild-type controls, DM2 and DM3 are spheroidal neuropils of
10 µm
diameters and are located in the mediodorsal surface of the lobe
(Fig. 1D,G). Va11m is crescent
in shape,
40 µm long and 10 µm wide, and is located on the
anterolateral surface, close to the nerve entry point
(Fig. 1J). In both
dockP1/dockP1 and
Pak6/Pak11 mutants, DM2, DM3 and VA11m are
severely mis-shapen or split into smaller structures that scattered randomly
in the AL (Fig. 1). In
dock, 42% of DM2 (n=26) and 75% of DM3 (n=36) are
ectopically located or splinter into smaller structures, scattered in the
neuropil (Fig. 1E,H). Although
the integrity and position of VA11m remain relatively unchanged in both
dock and Pak mutants, it is enlarged and appears to extend
into the domains of surrounding glomeruli. In 95% of dock
(n=20) and 95% of Pak (n=18) ALs it is seen to
engulf the adjacent VA1d glomerulus completely
(Fig. 1K,L). There does not
appear to be any consistent pattern in the distribution of the ectopic
glomeruli, which are scattered randomly and differ from one AL to another. As
assessed by the randomness in the positions of DM2 and DM3, and the aberrant
structure of VA1lm, the phenotype of Pak is indistinguishable from
that of dock, although defects occur at a slightly lower penetrance
(for example, 66% of DM3, n=18;
Fig. 1I).
dock and Pak function autonomously in olfactory
neurons
The severe defects in glomerular development of dock and
Pak mutants lead to the critical question of the focus of
dock and Pak actions in the developing AL. The precise
formation of the olfactory map requires multiple interactions between
ingrowing olfactory fibers and major cell types of the AL, such as the PNs and
AL glia (Oland and Tolbert,
1996). In theory, dock and Pak may act in any of
these cells to direct the wiring of the AL.
Staining with the anti-Dock antibody showed that Dock is highly expressed
in the dendrites and axons of ONs during the period of axon pathfinding to the
AL (see supplemental figures at
http://dev.biologists.org/supplemental/).
Staining with the anti-Pak antibody showed that Pak is similarly localized to
ON axons (data not shown), although the lack of a Pak protein null mutation
prevents us from assessing the specificity of the staining. The presence of
Dock and Pak proteins in ON axons is consistent with a role in ON axon
guidance. To test the notion that the proteins are needed in ONs, we asked if
removal of dock and Pak functions specifically from ONs
would disrupt their glomerular targeting. We found that the
ey-FLP/FRT method of site-specific recombination
(Newsome et al., 2000a)
induced the formation of large patches of homozygous tissues in the
eye-antennal disc but not in the brain (see supplemental figures at
http://dev.biologists.org/supplemental/).
We used the ey-FLP/FRT system to test whether dock and
Pak are required in the antenna for the precise targeting of ON
axons. Clones of wild-type, dockP1 and
Pak16 mutant tissues were induced in the antennae of
animals carrying either Or47a-Gal4 or Or47b-Gal4 transgenes
(see Fig. 2 legend for the
respective genotypes). The mosaic animals were allowed to develop to adulthood
and their brains were stained with nc82 mAb and anti-GFP to probe glomerular
development. In animals with wild-type antennae, the AL neuropil is subdivided
into well-defined glomeruli and ON axons converged precisely on their target
glomeruli (Fig. 2A). By
contrast, in animals with dockP1/dockP1 or
Pak16/Pak16 antennae, the ALs are severely
mis-shapen and aglomerular, and ON axons terminated in ectopic locations
(Fig. 2B,C). We conclude from
these observations that dock and Pak function within the
antennae to regulate the precise targeting of ON axons.
|
The mosaic experiments do not preclude an additional requirement for
dock and Pak in the brain or in nonneuronal cells of the
antenna. To test the hypothesis that dock and Pak function
specifically in ONs for AL development, we asked if the expression of
wild-type dock and Pak cDNAs in ONs would rescue the
dock and Pak mutant phenotypes. The enhancer trap line
SG18.1-Gal4 (Jhaveri et al.,
2000) was chosen to drive the expression of the cDNAs because we
observed that it is preferentially expressed in a subpopulation of ONs with
little expression in the brain. Examination of antennae from
SG18.1-Gal4/UAS-mCD8::GFP animals at 30 hours after puparium
formation (hAPF) showed that GFP is expressed only in cells with a bipolar
morphology characteristic of ONs (Fig.
2D). Examination of the brain in pupae at 30 and 55 hAPF, and in
the adult, showed that GFP is specifically localized to the nerve layer of the
AL and in numerous glomeruli, with little staining elsewhere
(Fig. 2E,
Fig. 3A). Removal of both the
antennae and maxillary palps abolished GFP expression in the ALs, showing that
the AL staining is contributed by olfactory afferents from these two organs
(Fig. 2F). Hence,
SG18.1-Gal4 is expressed preferentially in ONs of the antenna and the
maxillary palp. Expression of dock and Pak cDNAs under the
control of SG18.1-Gal4 strongly rescued the glomerular structures of
the AL (Fig. 2G-J). We
monitored four landmark glomeruli (VA1d, VA6, DA4 and DM6) as an indicator of
normal AL development (Fig.
2K). Expression of cDNAs under the control of SG18.1-Gal4
increased the average frequency of distinctly identifiable glomeruli from 14%
to 85% in dockP1/dockP1 ALs and 35% to 94% in
Pak6/Pak11 ALs. Although the rescue was strong,
we noticed that not all glomeruli were restored. The incomplete rescue is
probably due to the lack of SG18.1-Gal4 expression in some glomeruli
(Fig. 2E). Nonetheless, the
ability of SG18.1-Gal4 to support the substantial rescue of the
mutant phenotypes indicates that it is suitably active during olfactory axon
pathfinding. We conclude from these experiments that dock and
Pak genes are specifically required in the ONs to direct the guidance
of the ON axons.
|
dock and Pak regulate the precise pathfinding of
olfactory axons
We infer from the ectopic glomeruli that ON axons fail to project to their
correct destinations in dock and Pak mutants. To assess
whether ON axons are misprojecting in dock and Pak mutants,
we probed fiber pathways from the antenna to the AL. During development, ON
axons leave the antenna and travel to the AL between 20 and 50 hAPF
(Jhaveri et al., 2000
). Axon
pathways in the pupal antenna were visualized by staining 30 hAPF antennae
with 22C10, a mAb against Futsch (see Fig.
6A), while pathways in the adult AL were labeled with either the
mCD8::GFP or GAP::GFP fusion proteins (encoded by UAS-mCD8::GFP or
UAS-GAP::GFP) expressed under the control of various Gal4
drivers. Axon trajectories were examined with standard fluorescence microscopy
or after three-dimensional (3D) reconstructions of 2D confocal sections.
Fig. 3A shows a representative
reconstruction of a wild-type adult AL labeled by the SG18.1-Gal4
driver, which is expressed in coeloconic and trichoid sensilla
(Jhaveri and Rodrigues, 2002
).
The overall pattern of ON projection in the AL is bilaterally symmetric and
relatively invariant between individuals (see
Fig. 3B1 for an
interpretive drawing). GFP-expressing fibers extend through the antennal nerve
and enter the AL at its anterolateral point (close to VA1lm). Thereafter, the
axons radiate over the AL surface in characteristic tracks within a fibrous
nerve layer, to target their cognate glomeruli. Contralateral axons course
through a prominent commissure that connects the opposite ALs. That the
individual ON classes project in stereotyped pathways was confirmed by
labeling ONs with the Or22a-Gal4, Or47a-Gal4 and Or47b-Gal4
drivers. In confocal reconstructions, Or47a axons can be seen to take
relatively straight paths from the nerve entry point to DM3, their target
glomerulus (Fig. 3E,F1).
Examination of single Or47a axons (n=83) using the MARCM
technique (Lee and Luo, 1999
)
showed that each axon projects determinately to DM3, where it splits into an
ipsilateral branch, that terminates in DM3, and a contralateral branch, that
projects across the commissure (Fig.
4A,B). Axons appear to extend directly to DM3 without making
substantial changes in direction. Many fibers appear to project individually,
although a number of fibers also merge into fascicles as they converge upon
their targets (Fig. 3E).
Or47a axons always remain in the nerve layer throughout the entire
trajectory until in the vicinity of their target, whereupon the converging
fibers enter the lobe and terminate precisely on DM3. Or47b axons,
however, terminate immediately upon disembarking from the nerve, establishing
the crescent shape VA1lm (Fig.
3I,J1). A distinct fascicle issues from the medial edge
of VA1lm carrying collaterals between the glomerulus and the commissure.
Single-axon analysis (n=38) showed that the contralateral axons
project in relatively straight paths, seldom straying beyond the confines of a
narrow zone, between VA1lm and the commissure
(Fig. 4E,F). Our analyses show
that in the wild type, olfactory axons make bilaterally symmetric and
stereotyped patterns of projections to their glomeruli in the AL.
|
|
|
In antennae of dockP1/dockP1 and Pak4/Pak6 mutants at 30 hAPF, cells exhibiting a bipolar morphology can be seen, projecting their axons out of the antenna in distinct fascicles, a pattern indistinguishable from the wild type (Fig. 6A-C). However, once in the AL, the pathways are clearly abnormal. In both dock and Pak mutants, instead of forming characteristic tracks, SG18.1-Gal4-expressing fibers interweave to form a dense mat (Fig. 3B2-D). In 22% (n=32) of the dockP1/dockP1 brains, ON axons extend aberrantly to dorsal brain regions (Fig. 3C, right AL). Disruption in the overall projection pattern is reflected in the trajectories of the individual ON subtypes. In 3D confocal reconstruction, Or47a axons can be seen to deviate from their stereotyped pathways from the outset, veering to distant part of the AL, including even the core, in chaotic, meandering trajectories (Fig. 3F2). Visualization of single Or47a axons in dockP1/dockP1 mutants showed that misrouting affect both the ipsilateral and contralateral branches of an axon (Fig. 4C,D). In 21% of the cases (n=45), both branches remain in the ipsilateral AL. Most of the branches (64%, n=45) ultimately fail to reach their destination, forming mis-shapen glomeruli in ectopic locations. Interestingly, in 9% (n=45) of the cases, three axon branches were observed. However, the extra branch is usually short and not associated with ectopic glomeruli. Although Or47b axons terminate in a single large glomerulus as in wild type, it is strongly mis-shapen in dock and Pak mutants (Fig. 3J2). Furthermore, the contralaterally projecting Or47b axons are severely defasciculated, projecting through a wide area of the AL surface. Examination of single Or47b neurons showed that whereas ipsilateral axons terminate normally in VA1lm, contralateral axons are frequently misrouted, either projecting to dorsal brain regions or stopping short of the VA1lm glomerulus (Fig. 4G,H). Our analyses of the dock and Pak mutant phenotypes therefore show that dock and Pak are not necessary either for the outgrowth of olfactory axons or for their projection through the antennal nerve. Instead, dock and Pak function primarily in the guidance of ON axon within the AL to steer the sensory fibers precisely to their cognate glomeruli.
Interaction between Dock and Pak is critical for proper AL
development
The similarity in dock and Pak olfactory connectivity
phenotypes suggests that these genes might function in a signaling cascade to
regulate the targeting of ON axons. It has previously been shown that Dock and
Pak interacts through the N-terminal PXXP motif of Pak and the second SH3
domain of Dock (Hing et al.,
1999). We now observed that the P9L mutation
(Pak4), which affects the N-terminal PXXP motif of Pak and
abolishes its ability to bind to Dock, strongly disrupts ON axon pathfinding
(Fig. 3H). To evaluate the
hypothesis that Dock-Pak interaction is critical for ON axon guidance, we
examined the requirements of the individual Dock domains for AL development.
Expression of wild-type UAS-dock cDNA
(Rao and Zipursky, 1998
) under
the control of SG18.1-Gal4 significantly rescued the
dockP1/dockP1 aglomerular phenotype (compare
Fig. 5A with 5B). Although
mutations that disrupt the first SH3 domain, the third SH3 domain or the SH2
domain do not affect the ability of dock to rescue the mutant
phenotype (Fig. 5C,E,F), a
mutation that disrupts the second SH3 domain completely abolished
dock activity (Fig.
5D). The requirement of both the N-terminal PXXP motif of Pak and
the second SH3 domain of Dock suggests that physical interaction between Dock
and Pak is necessary for proper AL development. To obtain evidence of a
functional relationship between dock and Pak we tested for
genetic interactions between the genes. First, we asked if simultaneous
reduction of dock and Pak functions by half would disrupt AL
development. The ALs of dockP1/+;
Pak4/+ animals exhibit normal axon trajectories and glomerular
subdivisions similar to those of wild-type or heterozygous animals (see
supplemental figures at
http://dev.biologists.org/supplemental/).
However, the absence of genetic interaction in the compound heterozygotes
should not be taken as evidence against dock and Pak
functioning in the same signaling cascade. It is still possible that the
decreased levels of dock and Pak are sufficient for normal
functioning of the signaling pathway. We also asked if expression of the
constitutively active Pakmyr
(Hing et al., 1999
) in the
dock mutant would correct the mutant phenotype. However, expression
of UAS-Pakmyr with SG18.1-Gal4 in wild type
strongly disrupted the AL structure (L.-H. A and H. H., unpublished). The
strong gain-of-function phenotype makes its use in genetic epistasis studies
unfeasible.
|
dock and Pak do not function in antennal neuron
differentiation
As dock and Pak are autonomously required in ONs, one
explanation for defective targeting of their axons is that cell fate
specification of the neurons is disrupted. To explore this possibility, we
examined in detail the critical steps of ON differentiation in both mutants.
By monitoring ON development at various stages, we demonstrate that ONs
differentiate normally in dockP1/dockP1 and
Pak6/Pak11 homozygotes. First, antennal discs
of dock and Pak mutants show lozenge gene
expression in three semi-elliptical domains similar to wild type (see
supplemental figures at
http://dev.biologists.org/supplemental/).
Second, in nascent antennae derived from 30 hAPF mutant pupae, ONs express
both the ELAV and Futsch antigens (Fig.
6A-C). Furthermore, axons extend out of the antenna normally.
Third, in the mutant antennae, Or22a, Or47a and Or47b genes
are expressed by characteristic numbers of cells that are scattered randomly
within circumscribed domains as in wild type
(Table 2;
Fig. 6G-I; see supplemental
figures at
http://dev.biologists.org/supplemental/)
(Vosshall et al., 1999;
Vosshall et al., 2000
).
Fourth, the three olfactory sensilla types (basiconic, trichoid and
coeloconic) are found on the mutant antennae and have similar morphology,
numbers and distribution as in wild type
(Table 2,
Fig. 6D-F; see supplemental
figures at
http://dev.biologists.org/supplemental/)
(Shanbhag et al., 1999
).
Examination of indigenous cells of the AL, such as the PNs and the AL glia
(see supplemental figures at
http://dev.biologists.org/supplemental/),
showed that these cells also differentiate normally in the dock and
Pak mutants. However, in the mutants, the dendritic arborization of
PNs are more diffused and the number of glia processes are somewhat reduced.
Thus, morphogenetic changes of the cells that accompany normal glomerular
development fail to occur properly when the dock or Pak
genes are disrupted.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ONs of the antennae and maxillary palps undergo terminal differentiation
during early metamorphosis and become predestined to express particular Or
genes and synapse in specific glomeruli
(Jhaveri et al., 2000;
Vosshall et al., 1999
).
Between 20 and
50 hAPF, their axons leave the nascent antenna in
fascicles and enter the AL in search of their targets. PNs, however, acquire
their cell fates, which predetermine their glomerular choice, during larval
development (Jefferis et al.,
2001
). During early pupal development their dendrites enter the AL
and become precisely paired with ON axons in specific glomeruli. Thus, ONs
expressing a given Or gene rendezvous with PNs of a particular identity within
a topographically defined glomerulus in the AL.
We find that in the wild type, olfactory axons take stereotyped paths on
the surface of the AL to converge on their cognate glomeruli. Detailed
characterization of the axon trajectories, using Gal4 drivers
expressed in different subclasses of ONs shows that, upon arrival at the
anterolateral point of the AL, afferents project directly, with little
sidetracking to their postsynaptic targets. As in the mouse and moth, these
axon pathways are bilaterally symmetric and invariant from AL to AL
(Mombaerts et al., 1996;
Oland et al., 1998
;
Wang et al., 1998
). How is
this precise wiring pattern formed during development? In one model, each ON
initially sends collaterals to multiple glomeruli and then withdraws the
inappropriate branches in a process requiring odorant-evoked activity.
Alternatively, the invariant pattern of connections is the result of directed
axon migrations in response to spatially restricted pathfinding cues in the
developing AL. A definitive answer to this question will require developmental
study or direct observation of the extending axons. However, several
observations are consistent with the notion that olfactory axons navigate
directly to their cognate glomeruli. First, a temporal lag between early axon
pathfinding and subsequent Or gene expression
(Clyne et al., 1999
) indicates
that an odorant-evoked activity is unlikely to play an important role. Indeed,
activity is neither required for formation nor maintenance of the olfactory
map in mouse and moth (Belluscio et al.,
1998
; Lin et al.,
2000
; Oland et al.,
1996
). Second, and importantly, our finding that the growth cone
guidance genes, dock and Pak, are needed for development of
the olfactory map, provides strong evidence that directed axon migration plays
a key role in the matching of ON axons with their correct glomeruli. Directed
navigation of olfactory axons to their targets is also observed in zebrafish
and moth (Dynes and Ngai, 1998
;
Oland et al., 1998
).
In dock and Pak mutants, the stereotyped connectivity of
AL neuropil is severely disrupted, leading to an aglomerular phenotype. We
present three pieces of evidence indicating that dock and
Pak function in ONs. First, antibody staining shows that Dock and Pak
proteins are expressed in antennal axons during the period in which they are
projecting to the brain. Consistent with their requirements in ONs, removal of
dock and Pak activities from only the antennae results in
ectopic targeting of olfactory axons. Finally, expression of dock and
Pak cDNAs specifically in ONs in otherwise mutant animals leads to
strong rescue of the mutant AL phenotype. We noticed that although numerous
glomeruli were restored upon the expression of the wild-type cDNAs, some
glomeruli were not. We believe that the incomplete rescue is due to the
expression of SG18.1-Gal4 in only a subset of all the ONs. However,
it is also possible that the partial rescue reflects an additional requirement
of dock and Pak functions in the brain. A recent study
indicates that ONs may be divided into different classes based on the timing
of their projections (Jhaveri and
Rodrigues, 2002). We did not determine further if dock
and Pak are required in all ONs or in only a specific subset.
Although dock and Pak are specifically required in ONs, our
finding of nonautonomous effects on the morphogenetic changes of the PNs and
AL glia is in accord with earlier studies in which ONs were physically or
genetically ablated (Graziadei and
Monti-Graziadei, 1992
;
Hildebrand et al., 1979
;
Stocker and Gendre, 1988
). Our
data therefore show that proper termination of ON axons is also an important
step in the sculpting of the AL neuropil into distinct glomeruli.
We provide evidence that the disruption in AL development in dock
and Pak mutants is not an indirect effect of aberrant cell-fate
determination or axonogenesis. By contrast, we observed that the precise
targeting of ON axons is severely disrupted in dock and Pak
mutants. To identify the cause of the mistargeting, we examined the axon
pathways of individual ON classes (Or47a, and Or47b) at the
single-cell level. Although an additional short branch was observed in 9% of
dock mutant neurons (n=45), the most striking defect
observed in single-cell clones (64%, n=45) is the chaotic
trajectories exhibited by both the ipsilateral and contralateral axons of the
ONs. We conclude that the primary function of dock and Pak
in ONs is axon pathfinding, to steer ON axons precisely to their target
glomeruli. In mouse, mutations in the odorant receptor genes abolish the
ability of olfactory axons to pathfind in the anteroposterior axis without
affecting their migration in the dorsoventral axis, leading to the proposal
that odorant receptors participate in the recognition of only anteroposterior
guidance cues (O'Leary et al.,
1999; Wang et al.,
1998
). However, after examining several hundred ALs for each
dock and Pak mutant, we did not observe any consistent
patterns in the mistargeting of ON axons. We did observe that the ON classes
are affected to different degrees by the loss of dock and
Pak activities. Although Or22a and Or47a axons
terminate in numerous ectopic glomeruli, Or47b axons terminate in a
single glomerulus, albeit mis-shapen, in the approximate position of the
wild-type VA1lm. We currently do not know the reason for the differential
sensitivity of the ON subtypes to the loss of dock and Pak
functions. One possibility is that Or47b axons, which are among the
first axons to enter the AL, are confronted with fewer developing glomeruli
(Jhaveri et al., 2000
) and
hence fewer guidance choices than Or22a and Or47a axons that
enter the AL later. Alternatively, Or47b axons may have less need for
dock- and Pak-mediated navigational functions because VA1lm
is located near the nerve entry point. Indeed, while the Or47b
ipsilateral axons frequently terminate accurately on VA1lm, the contralateral
axons, which have to project across the entire AL surface, are often
misrouted. In contrast to the severe projection defects in the AL, the
migration of dock and Pak mutant axons through the antennal
nerve takes place normally. It is possible that the lack of requirement of
dock and Pak functions during this phase of axon growth
reflects a different guidance mechanism in the antennal nerve.
The observation that the ON axon trajectories are severely disrupted in
dock and Pak mutants suggests that the genes may mediate the
detection or response of the growth cones to guidance cues in the environment.
Our results indicate that in these events, dock and Pak are
very likely to act in a signaling pathway. First, loss of either dock
or Pak functions results in olfactory connectivity phenotypes that
are indistinguishable. Second, both dock or Pak function
autonomously in ONs. Third, mutations that disrupt the domains of Dock (second
SH3 domain) and Pak (N-terminal PXXP domain; Pak4), which
mediate interaction between the two proteins
(Hing et al., 1999), disrupt
ON axon targeting. We therefore propose that Dock and Pak are part of a signal
transduction cascade that allows ONs to find and precisely pair with the
correct postsynaptic partners. Although severely disrupted, the guidance of ON
axons in dock and Pak mutants is not completely abolished,
indicating that other genes function to steer ON axons to their targets as
well.
As the Dock-Pak signaling pathway appears to govern the pathfinding of a
number of ON subtypes, it is unlikely to explain the specificity of ON
targeting. What transmembrane receptors might control the Dock-Pak signaling
pathway and direct the precise pairing between ONs and their postsynaptic
targets? Because ONs that target different glomeruli are interspersed in the
olfactory epithelium, thus precluding their tagging by simple gradients of
molecular cues, it is anticipated that the receptors are molecularly diverse.
A highly diverse receptor family is, of course, the odorant receptor family.
In mouse, odorant receptors themselves provide the specificity for target
selection (Mombaerts et al.,
1996; Wang et al.,
1998
). Odorant receptors are unlikely to play a guidance role in
Drosophila, however, as expression of these genes begin long after
axon migration has taken place (Clyne et
al., 1999
). Another family of diverse receptors that functions in
cell-cell adhesion and is expressed in synapses is the cadherin superfamily
(Yagi and Takeichi, 2000
). In
mouse, some members of the family, the CNRs, are expressed in the olfactory
bulb, indicating that cadherin family proteins may also play a role in
olfactory map development (Kohmura et al.,
1998
). Recently, in a biochemical screen for proteins that bind to
Dock, Zipursky and colleagues identified the immunoglobulin superfamily
transmembrane receptor Dscam (Schmucker et
al., 2000
). Interestingly, the expression of the Dscam
gene is regulated by a novel, combinatorial splicing mechanism, which allows
Dscam to encode up to 38,000 isoforms
(Schmucker et al., 2000
).
Because of the tremendous diversity in its gene products, Dscam has
been proposed to play a role in encoding synaptic specificity. Our discovery
that the Dock-Pak signaling pathway regulate the projection of ON axons to
their cognate glomeruli now presents us with a unique opportunity to begin to
assess the possible roles of these receptors in the development of the
Drosophila olfactory map.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashburner, M. (1989). Drosophila: A Laboratory Handbook. New York: Cold Spring Habor Laboratory Press.
Bagrodia, S. and Cerione, R. A. (1999). Pak to the future. Trends Cell Biol. 9, 350-355.[CrossRef][Medline]
Belluscio, L., Gold, G. H., Nemes, A. and Axel, R. (1998). Mice deficient in G(olf) are anosmic. Neuron 20,69 -81.[Medline]
Clemens, J. C., Ursuliak, Z., Clemens, K. K., Price, J. V. and
Dixon, J. E. (1996). A Drosophila protein-tyrosine
phosphatase associates with an adapter protein required for axonal guidance.
J. Biol. Chem. 271,17002
-17005.
Cloutier, J. F., Giger, R. J., Koentges, G., Dulac, C., Kolodkin, A. L. and Ginty, D. D. (2002). Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons. Neuron 33,877 -892.[Medline]
Clyne, P. J., Warr, C. G., Freeman, M. R., Lessing, D., Kim, J. and Carlson, J. R. (1999). A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22,327 -338.[Medline]
Daniels, R. H. and Bokoch, G. M. (1999). p21-activated protein kinase: a crucial component of morphological signaling? Trends Biochem. Sci. 24,350 -355.[CrossRef][Medline]
Dynes, J. L. and Ngai, J. (1998). Pathfinding of olfactory neuron axons to stereotyped glomerular targets revealed by dynamic imaging in living zebrafish embryos. Neuron 20,1081 -1091.[Medline]
Estes, P. S., Ho, G. L., Narayanan, R. and Ramaswami, M. (2000). Synaptic localization and restricted diffusion of a Drosophila neuronal synaptobrevingreen fluorescent protein chimera in vivo. J. Neurogenet. 13,233 -255.[Medline]
Firestein, S. (2001). How the olfactory system makes sense of scents. Nature 413,211 -218.[CrossRef][Medline]
Gao, Q. and Chess, A. (1999). Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics 60,31 -39.[CrossRef][Medline]
Gao, Q., Yuan, B. and Chess, A. (2000). Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci. 3, 780-785.[CrossRef][Medline]
Garrity, P. A., Rao, Y., Salecker, I., McGlade, J., Pawson, T. and Zipursky, S. L. (1996). Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein. Cell 85,639 -650.[Medline]
Graziadei, P. P. and Monti-Graziadei, A. G. (1992). The influence of the olfactory placode on the development of the telencephalon in Xenopus laevis. Neuroscience 46,617 -629.[CrossRef][Medline]
Hildebrand, J. G., Hall, L. M. and Osmond, B. C. (1979). Distribution of binding sites for 125I-labeled alpha-bungarotoxin in normal and deafferented antennal lobes of Manduca sexta. Proc. Natl. Acad. Sci. USA 76,499 -503.[Abstract]
Hing, H., Xiao, J., Harden, N., Lim, L. and Zipursky, S. L. (1999). Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila. Cell 97,853 -863.[Medline]
Jefferis, G. S., Marin, E. C., Stocker, R. F. and Luo, L. (2001). Target neuron prespecification in the olfactory map of Drosophila. Nature 414,204 -208.[Medline]
Jhaveri, D. and Rodrigues, V. (2002). Sensory
neurons of the Atonal lineage pioneer the formation of glomeruli within the
adult Drosophila olfactory lobe. Development
129,1251
-1260.
Jhaveri, D., Sen, A. and Rodrigues, V. (2000). Mechanisms underlying olfactory neuronal connectivity in Drosophila-the atonal lineage organizes the periphery while sensory neurons and glia pattern the olfactory lobe. Dev. Biol. 226, 73-87.[CrossRef][Medline]
Key, B. and St John, J. (2002). Axon navigation
in the mammalian primary olfactory pathway: where to next? Chem.
Senses 27,245
-260.
Knoll, B., Zarbalis, K., Wurst, W. and Drescher, U.
(2001). A role for the EphA family in the topographic targeting
of vomeronasal axons. Development
128,895
-906.
Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R., Watanabe, M., Ishii, H., Yasuda, M., Mishina, M. and Yagi, T. (1998). Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20,1137 -1151.[Medline]
Laissue, P. P., Reiter, C., Hiesinger, P. R., Halter, S., Fischbach, K. F. and Stocker, R. F. (1999). Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster. J. Comp. Neurol. 405,543 -552.[CrossRef][Medline]
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible neurotechnique cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[Medline]
Li, W., Fan, J. and Woodley, D. T. (2001). Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton. Oncogene 20,6403 -6417.[CrossRef][Medline]
Lin, D. M., Wang, F., Lowe, G., Gold, G. H., Axel, R., Ngai, J. and Brunet, L. (2000). Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity. Neuron 26,69 -80.[Medline]
Mombaerts, P. (2001). How smell develops. Nat. Neurosci. 4 Suppl,1192 -1198.[CrossRef][Medline]
Mombaerts, P., Wang, F., Dulac, C., Chao, S. K., Nemes, A., Mendelsohn, M., Edmondson, J. and Axel, R. (1996). Visualizing an olfactory sensory map. Cell 87,675 -686.[Medline]
Newsome, T. P., Asling, B. and Dickson, B. J.
(2000a). Analysis of Drosophila photoreceptor axon guidance in
eye-specific mosaics. Development
127,851
-860.
Newsome, T. P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A. and Dickson, B. J. (2000b). Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell 101,283 -294.[Medline]
O'Leary, D. D., Yates, P. A. and McLaughlin, T. (1999). Molecular development of sensory maps: representing sights and smells in the brain. Cell 96,255 -269.[Medline]
Oland, L. A. and Tolbert, L. P. (1996). Multiple factors shape development of olfactory glomeruli: insights from an insect model system. J. Neurobiol. 30, 92-109.[CrossRef][Medline]
Oland, L. A., Pott, W. M., Bukhman, G., Sun, X. J. and Tolbert, L. P. (1996). Activity blockade does not prevent the construction of olfactory glomeruli in the moth Manduca sexta. Int. J. Dev. Neurosci. 14,983 -996.[Medline]
Oland, L. A., Pott, W. M., Higgins, M. R. and Tolbert, L. P. (1998). Targeted ingrowth and glial relationships of olfactory receptor axons in the primary olfactory pathway of an insect. J. Comp. Neurol. 398,119 -138.[CrossRef][Medline]
Rao, Y. and Zipursky, S. L. (1998). Domain
requirements for the Dock adapter protein in growth- cone signaling.
Proc. Natl. Acad. Sci. USA
95,2077
-2082.
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.[Medline]
Schwarting, G. A., Kostek, C., Ahmad, N., Dibble, C., Pays, L.
and Puschel, A. W. (2000). Semaphorin 3A is required for
guidance of olfactory axons in mice. J. Neurosci.
20,7691
-7697.
Scott, K., Brady, R., Jr, Cravchik, A., Morozov, P., Rzhetsky, A., Zuker, C. and Axel, R. (2001). A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104,661 -673.[Medline]
Sells, M. A., Boyd, J. T. and Chernoff, J.
(1999). p21-activated kinase 1 (Pak1) regulates cell motility in
mammalian fibroblasts. J. Cell Biol.
145,837
-849.
Shanbhag, S. R., Muller, B. and Steinbrecht, R. A. (1999). Atlas of olfactory organs of Drosophila melanogaster. 1. Types, external organization, innervation and distribution of olfactory sensilla. Int. J. Insect Morphol. Embryol. 28,377 -397.[CrossRef][Medline]
Stocker, R. F. and Gendre, N. (1988). Peripheral and central nervous effects of lozenge3: a Drosophila mutant lacking basiconic antennal sensilla. Dev. Biol. 127, 12-24.[Medline]
Stocker, R. F., Lienhard, M. C., Borst, A. and Fischbach, K. F. (1990). Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Res. 262, 9-34.[Medline]
Stocker, R. F., Heimbeck, G., Gendre, N. and de Belle, J. S. (1997). Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J. Neurobiol. 32,443 -456.[CrossRef][Medline]
Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96,725 -736.[Medline]
Vosshall, L. B., Wong, A. M. and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102,147 -159.[Medline]
Wang, F., Nemes, A., Mendelsohn, M. and Axel, R. (1998). Odorant receptors govern the formation of a precise topographic map. Cell 93, 47-60.[Medline]
Yagi, T. and Takeichi, M. (2000). Cadherin
superfamily genes: functions, genomic organization, and neurologic diversity.
Genes Dev. 14,1169
-1180.