1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
2 Institute of Molecular and Cellular Biosciences, The University of Tokyo,
Tokyo 113-0032, Japan
* Author for correspondence (e-mail: lluo{at}stanford.edu)
Accepted 9 December 2004
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SUMMARY |
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Key words: Olfaction, Projection neuron, Metamorphosis, Pruning, Steroid hormone, TGFß signaling, Antennal lobe, Mushroom body, Lateral horn, Drosophila
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Introduction |
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One of the best-studied examples of neuronal reorganization in an insect
central nervous system is the neuron of Drosophila mushroom
bodies (MBs) (Technau and Heisenberg,
1982
; Armstrong et al.,
1998
; Lee et al.,
1999
; Lee et al.,
2000
; Watts et al.,
2003
; Watts et al.,
2004
; Zheng et al.,
2003
; Awasaki and Ito,
2004
). MB
neurons are born during embryonic
(Armstrong et al., 1998
) and
early larval stages (Lee et al.,
1999
). They send dendrites into the MB calyx and axons into larval
medial and dorsal MB axon lobes. During early metamorphosis,
neurons
prune their larva-specific dendrites and axon branches before re-extending
adult-specific processes (Lee et al.,
1999
). What happens to their synaptic partners while MB
neurons reorganize their dendrites and axons? In this study, we show that a
subset of olfactory projection neurons the major presynaptic partners
of MB
neurons are also morphologically differentiated to
function in both larva and adult. We also show that the reorganization of
these neurons during metamorphosis is independently controlled by some of the
same molecular mechanisms as that of the MB
neurons.
In the adult fly, odors are detected by olfactory receptors (ORs) on the
dendrites of about 1300 olfactory receptor neurons (ORNs) in the antennae and
maxillary palps. In general, each ORN appears to express one of 45
possible OR types (Clyne et al.,
1999
; Gao and Chess,
1999
; Vosshall et al.,
1999
), and the axons of all ORNs expressing a given OR converge to
one of
45 stereotypical glomeruli in the antennal lobe (AL), the
equivalent of the mammalian olfactory bulb
(Gao et al., 2000
;
Vosshall et al., 2000
). From
there, 150-200 projection neurons (PNs) relay olfactory activity to higher
brain centers, the MB calyx and the lateral horn (LH) of the protocerebrum
(Stocker, 1994
). Systematic
clonal analysis using the MARCM method
(Lee and Luo, 1999
) to label
single and clonally related clusters of PNs that express the GAL4 driver GH146
(Stocker et al., 1997
)
revealed that these PNs are prespecified by lineage and birth order to
innervate particular glomeruli in the adult AL
(Jefferis et al., 2001
).
Moreover, each glomerular class of PNs exhibits a characteristic axon
branching pattern in the LH, suggesting stereotyped targets in at least one
higher olfactory center (Marin et al.,
2002
; Wong et al.,
2002
).
The Drosophila larval olfactory system is much smaller and simpler
by comparison (Cobb and Domain,
2000; Heimbeck et al.,
1999
), shown to consist of only 21 ORNs in the dorsal organ
(Singh and Singh, 1984
) and
believed to include
50 PNs relaying information to the larval MB and LH
(Python and Stocker, 2002
;
Stocker, 1994
). Developmental
analysis has shown that the PNs born during larval stages exhibit only a
single unbranched process from the cell body to the MB calyx until early
metamorphosis, when dendrites and axon terminal branches start to elaborate
(Jefferis et al., 2004
). Thus,
larval-born PNs do not participate in the larval olfactory circuit.
What, then, is the origin of the relay interneurons that connect the larval
AL to higher olfactory centers? Do they contribute to the adult olfactory
system as well? Here we show that, in contrast to the larval-born PNs, PNs
generated during embryogenesis exhibit morphologically differentiated
dendrites and axons in both larva and adult. These neurons prune their
processes locally during the first few hours of metamorphosis and later
re-extend them to innervate developing adult structures. This pruning process
is regulated by ecdysone and TGFß signaling, as has been demonstrated
previously for MB neurons (Lee et
al., 2000
; Zheng et al.,
2003
). Thus, developmentally programmed remodeling allows these
embryonic-born PNs to participate in two distinct olfactory circuits at two
different stages in the Drosophila life cycle.
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Materials and methods |
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All embryos were collected on grape juice agar plates at 25°C. For embryonic heatshock, embryos were stored at 16°C before and until 24 hours after the heatshock; the temperature was then raised stepwise through 18°C, 20°C, and finally 25°C to prevent unintentional hs-FLP induction. Wandering third instar larvae were collected and dissected immediately. White pre-pupae were collected and then aged at 25°C for the desired number of hours prior to dissection.
Dissection and immunohistochemistry
Adult brain dissection and immunostaining were according to protocols
described previously (Jefferis et al.,
2001). Larval and pupal brains were dissected and stained using a
protocol modified from Python and Stocker
(Python and Stocker, 2002
).
Briefly, brains were dissected into phosphate-buffered saline (PBS) with 0.2%
Triton-X and fixed in 4% paraformaldehyde for 1-2 hours at 4°C. Brains
were then washed three times in PBS-Triton and blocked in 3% normal goat serum
at room temperature, and incubated overnight at 4°C with rat monoclonal
anti-mCD8
subunit (Caltag, Burlingame, California) at 1:100 and either
mouse monoclonal nc82 (kind gift of E. Buchner, University of Wuerzberg) at
1:20, rabbit polyclonal anti-synaptotagmin (kind gift of H. Bellen, Baylor
College of Medicine) at 1:1000, or mouse monoclonal anti-EcRB1 (AD4.4, kind
gift of C. Thummel, University of Utah) at 1:5000. Brains were again washed
three times in PBS-Triton at RT and incubated overnight at 4°C with
Alexa-488 goat anti-rat IgG at 1:300 and Alexa-568 goat anti-mouse IgG or
Alexa-568 goat anti-rabbit IgG at 1:300 (Molecular Probes, Eugene, OR). Brains
were washed, equilibrated using the Slow-Fade Light Anti-Fade Kit (Molecular
Probes, Eugene, OR), and whole mounted on glass slides.
Confocal and electron microscopy
Stacks of optical confocal sections at 1-2 µm spacing were obtained with
a Bio-Rad MRC 1024 laser-scanning confocal microscope using the Laser Sharp
image collection program. Raw images were processed using the freeware program
ImageJ and the commercial software Adobe Photoshop. Presented images are
either single slices or flattened confocal stacks, as indicated. Electron
microscopy studies were performed as previously described
(Watts et al., 2004).
Clonal analysis
We observed single-cell clones innervating adult AL glomeruli VL2p+, DP1m,
DP1m+DM4, VA2, VA6, VL2a, VL2a+DC1, DC1, VA7l, DA4, DM3, DM7, VM3, DL6 and
either D or a glomerulus just posterior. In addition, we saw DL2d and
DP1m-independent DM4 labeling in Nb clones, although not as yet in any
single-cell clones. From this available set, we selected eight landmark
glomeruli (DP1m, VL2p, VA6, VA2, DL5, DM3, VM3 and DL6) for the neuroblast
clone birth order study.
In the analysis of mutants (usp or babo), it is in theory possible that some of the MARCM phenotypes were caused by non-autonomous effects of neurons that were homozygous mutant but not labeled by GAL4-GH146. We think this is unlikely because the frequency of mitotic recombination induced by heatshocking during embryogenesis is extremely low (1-2 orders of magnitude lower than heatshocking in larvae). Given the penetrance of the phenotypes, it is unlikely that coincident unlabeled homozygous clones could account for the defects observed.
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Results |
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By comparing the specific glomeruli innervated in each partial anterodorsal
neuroblast clone generated by heatshock at different times during
embryogenesis (n=73), we ascertained that: (1) these embryonic-born
PNs were generated in the order DP1m, VL2p, VA6, VA2, DL5, DM3, VM3 and
finally DL6 (Table 1); and (2)
every clone labeled by embryonic heatshock included all of the larval-born
anterodorsal PNs analyzed in our previous study, indicating that both PN
subsets originate from the same neuroblast. Upon generation of the DL6 PN(s),
the anterodorsal neuroblast apparently arrests, only producing additional
projection neurons later in larval life (as indicated by heatshock-induced
labeling of just a single anterodorsal glomerular class, DL1, until about 36
hours after larval hatching) (Jefferis et
al., 2001).
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Embryonic-born projection neurons participate in the larval olfactory system
Given their early origin, these GH146-positive embryonic-born PNs may
participate in the larval olfactory circuit as well. Indeed, examining third
instar larval brains reveals that GH146 is strongly expressed in presumptive
projection neurons that appear to innervate the larval AL and to send axons up
to the MB calyx and larval equivalent of the adult LH
(Fig. 2A)
(Stocker et al., 1997). These
projections appear to be contributed by about 16 to 18 clustered neurons that
are presumably derived from the anterodorsal neuroblast.
|
Several lines of evidence suggest that the embryonic-born PNs observed in the larval olfactory system are the same cells as the PNs that contribute to the much larger and more complex adult circuit. First, the frequencies of labeled single-cell clones are comparable between the two stages, arguing against the possibilities that embryonic-born PNs are either dying off during metamorphosis or remaining quiescent and undetected through larval life. Second, the numbers of GH146-positive PNs observed at the time of puparium formation and in the adult are similar. Third, and most importantly, each embryonic-born PN undergoes characteristic morphological changes during metamorphosis, as described in detail below. Therefore, for the remainder of this paper, we will refer to the PNs labeled by embryonic heatshock as persistent projection neurons (PPNs), whether we are examining them in larva, pupa or adult. However, at this point, our methods do not allow us to correlate specific glomerular classes in larva with those observed later in adulthood.
Persistent projection neurons prune processes locally during early metamorphosis
How do PPNs participate in both larval and adult olfactory circuits? The
larval and adult olfactory systems differ considerably in size and complexity,
and at least some of the presumed synaptic partners of the PPNs, the MB
neurons, are known to prune their processes during metamorphosis
(Lee et al., 1999
). Do PPNs
remain essentially unaltered during metamorphosis, perhaps even providing cues
for the pathfinding and/or targeting of later-born neurons? Or, like the MB
neurons, do they prune their processes and then re-extend them to form
new connections in the adult brain?
We have attempted live imaging of projection neurons during metamorphosis in intact pupae using two-photon microscopy; unfortunately, the brain is obscured by the opaque pupal cuticle and subcuticular fat. Our attempts to culture pupal brains outside of the cuticle with its complicated hormonal milieu have not yet been successful (D. Berdnik, A. Goldstein and L. Luo, unpublished). However, using MARCM to label PPNs and then dissecting brains every 2 hours after puparium formation (APF) permitted a series of developmental snapshots. This timecourse revealed that each PPN prunes its dendrites and axon terminals locally during early metamorphosis, leaving the main axon trunk from the cell body to the MB calyx intact, and later extends new processes to target the developing AL, MB and LH of the adult brain (Fig. 3).
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PPN dendritic remodeling at larval AL
At the onset of puparium formation, PPN dendrites appear identical to those
found in third instar larvae, with a large dendritic density clearly visible
in the larval AL (score=1, Fig.
3A). But just 2 hours APF, dendrites in the larval AL appear
thinned (score=2, Fig. 3B), and
by 4 hours APF, they are mostly gone (score=3,
Fig. 3C). In the next 4-8
hours, the larval dendritic processes are eliminated
(Fig. 3I), while new filopodia
start to appear from the main process more dorsally, in the presumed
adult-specific AL (see Jefferis et al.,
2004). By 18 hours APF, new dendrites have only begun to target
specific regions of the newly forming adult-specific AL in two out of eight
samples examined. This implies that embryonic-born PNs initiate innervation of
the developing AL after the larval-born PNs, for which localized dendritic
outgrowth is already visible by 6-12 hours APF
(Jefferis et al., 2004
).
PPN axonal remodeling at MB and LH
The axons of PPNs exhibit similar morphological changes, although beginning
a little later in metamorphosis. For the first 2 hours APF, the majority of
axons appear unchanged (score=1, Fig.
3E). By 4 hours APF, the presumptive synaptic structures in the MB
calyx have become less dense, while the terminal branches in the larval LH are
being pruned (score=2, Fig.
3F). Axon pruning proceeds over the next 6 hours until the main
axon trunk terminates in the calyx, with no visible synaptic densities
remaining (score=4, Fig. 3H).
The remnants of PPNs in the calyx persist for a longer period of time compared
to AL or LH (score=3, Fig.
3I-K), possibly reflecting the time it takes to turn over large
presynaptic terminals there (see Fig.
5 below). Although fine filopodia sometimes can be seen to extend
from the end of the axon, no directed outgrowth is observed through 12 hours
APF. However, exuberant new processes re-extend into both the calyx and the LH
region by 18 hours APF in seven of eight samples examined.
|
To determine whether the latter possibility might indeed be the case, we made use of a second, more specific, projection neuron driver, GAL4-MZ612. This driver is strongly expressed by one PPN innervating glomerulus VA6 in the adult antennal lobe, previously identified by GH146 MARCM as an embryonic-born PN target (Fig. 1G). It also strongly labels one PN in the larval olfactory circuit, which innervates a medial glomerulus in the larval AL. MZ612-based MARCM experiments confirm that this neuron is labeled by inducing recombination during embryogenesis and that it persists to innervate VA6 in the adult.
Just like the GH146-positive PPNs, the MZ612-positive neuron prunes and then re-extends its processes in the first 18 hours after metamorphosis (Fig. 4A-H). Interestingly, the timecourse of reorganization appeared less variable for this homogeneous population of PPNs (Fig. 4I-K, compare with Fig. 3I-K). This supports the idea that at least some of the variation observed for the GH146-positive PNs was attributable to characteristic differences in pruning dynamics between glomerular classes.
|
Electron micrographs from wandering third instar larvae in which HRP::CD2
was driven by GH146 (Fig.
5A2,A3) or 201Y (Fig.
5B2,B3) gave complementary results supporting the following model.
Persistent projection neuron axon terminals appear as large presynaptic
boutons (2-4 µm in size; * in
Fig. 5A2,B2) that contain
synaptic vesicles apposing postsynaptic profiles (arrowheads in
Fig. 5A3,B3). These structures
are enriched in the peripheral calyx and probably correspond to
glomerulus-like structures seen with fluorescence microscopy
(Fig. 2E1-E3), analogous to the
`calycal glomerulus' described in adult calyx
(Yasuyama et al., 2002). Only
a subset of these large presynaptic boutons is labeled by GH146, consistent
with the fact that only a subset of PPNs are GH146-positive. MB
neuron
dendritic profiles are small and numerous (labeled in
Fig. 5B3), and are directly
postsynaptic to large profiles of presynaptic boutons presumed to be from PNs.
These experiments therefore provide direct morphological evidence that PPN
axons form functional synapses in the larval circuit with dendrites of MB
neurons.
At 6 hours APF, the peak of MB neuron dendrite pruning
(Watts et al., 2003
) and PPN
axon pruning (Fig. 3), electron
microscopy analyses of calyx labeled with PPNs
(Fig. 5A4,A5) or MB
neurons (Fig. 5B4,B5) revealed
that the large presynaptic boutons have been disassembled. Both PPN synaptic
terminals and MB
neuron dendritic profiles appear to be engulfed by
glia (Fig. 5A5,B5, open
arrowheads; glial profiles were identified by characteristic glycogen
granules), as has been shown recently for MB
neuron axons
(Watts et al., 2004
).
Requirement of ecdysone co-receptor and TGFß receptor for PPN pruning
Prior studies have used MB neurons as a model system to study the
molecular mechanisms of axon pruning.
neuron pruning depends on
cell-autonomous reception of the steroid hormone ecdysone; single neurons that
are homozygous mutant for the ecdysone co-receptor ultraspiracle
(usp) in an otherwise heterozygous brain fail to reorganize their
processes and retain both dorsal and medial axon lobes in the adult brain
(Lee et al., 2000
). In
addition,
neurons must upregulate the expression of ecdysone receptor
isoform B1 (EcRB1) prior to axon pruning
(Lee et al., 2000
). This
upregulation requires TGFß signaling, as MB
neurons that are
mutant for the TGFß/Activin Type I receptor baboon
(babo) or its downstream effector Dsmad do not upregulate
EcRB1 expression and consequently fail to prune
(Zheng et al., 2003
).
We asked whether a similar molecular pathway is utilized during PPN
reorganization. To ascertain whether the pruning of PPNs is also regulated by
ecdysone, we first examined EcRB1 expression patterns. At puparium formation,
only 20 of the 90 GH146+ projection neurons present were strongly
positive for EcRB1. These strongly stained PNs,
18 of which belonged to
the anterodorsal cluster, also had noticeably larger and brighter cell bodies
than surrounding PNs, which were probably immature larval-born PNs
(n=10/10). Single-cell MARCM clones generated by embryonic heatshock
were also strongly positive for EcRB1 at puparium formation
(Fig. 6A1, n=10/10).
Thus, we conclude that EcRB1 expression is highly expressed in PPNs at the
onset of metamorphosis.
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Consistent with the loss of EcRB1 expression, baboFd4
PPNs failed to reorganize their processes normally during the first few hours
of metamorphosis. For wild-type PPNs at 8 hours APF, approximately 95% of
dendrites, 80% of MB calyx processes, and 85% of LH processes are in the final
two stages of pruning (Fig.
6F). However, most of the embryonic-born
baboFd4 PPNs still retained dendrites and axons with
larval morphology at this time (Fig.
6F). Dense dendritic processes were visible in the larval AL for
100% of PN clones examined (Fig.
6D2,F), and only 14% of axon branches in the LH appeared to
resemble the final two stages of pruning
(Fig. 6E2,F). The degree of
pruning in the calyx was more difficult to estimate, due to the concurrent
degeneration of MB neuron dendrites and loss of glomerular
organization, but disappearance of synaptic boutons still seemed inhibited
(Fig. 6E2,F).
To confirm that this failure to prune resulted from loss of ecdysone signal
reception, we also used MARCM to label PPNs that were homozygous for a
well-characterized mutant allele (Lee et
al., 2000) of the ecdysone co-receptor, usp3.
At the wandering third instar stage, usp3 PPNs exhibit
normal morphology (Fig. 6B3,C3;
n=17/17), and, as expected, EcRB1 was expressed at wild-type levels
at the time of puparium formation (Fig.
6A3; n=10/10).
However, when we examined these brains at 8 hours APF, we observed a
significant defect in dendrite and axon pruning. In the majority of cases,
both dendritic densities in the location of the larval antennal lobe
(Fig. 6D3) and axon branches in
the MB calyx and LH had been retained (Fig.
6E3, quantified in Fig.
6F). Taken together, these mosaic experiments suggest that PPN
dendritic and axonal pruning require cell-autonomous function of EcRB1/USP, as
has been shown previously for MB neurons.
Adult phenotypes for PPNs defective in pruning
What are the consequences for the adult olfactory circuit when larval
circuits fail to prune? PPNs homozygous for usp3 or
baboFd4 that failed to prune their dendrites and axons
during metamorphosis allowed us to investigate this question.
When examined in adults, wild-type PPN dendrites were confined to a single glomerulus in the adult AL with the exception of the VL2p+ class (Fig. 1; Fig. 7A1). Dendrites of single-cell PPN clones homozygous for usp3 generally appeared to target glomeruli in the adult AL appropriate for PPNs; however, ectopic processes in additional areas of the AL (arrows in Fig. 7B1,B2; quantified in Fig. 7G), which could be interpreted as persisting larval dendrites, were often present. In a few cases, usp3 PPN dendrites were sparser and less specifically targeted to particular glomeruli, but still remained somewhat confined to certain regions of the AL (quantified in Fig. 7G). Likewise, whereas wild-type PPNs always exhibited terminal swellings on short side branches (Fig. 1; Fig. 7A2), about 40% of usp3 PPNs retained larval-like boutons directly on their main trunks in the MB calyx (arrowheads in Fig. 7B2,C2); however, they always had side branches with terminal swellings as well, implying that re-extension and adult-specific outgrowth were not completely impaired. In addition, the main axon trunk often diverted conspicuously from the inner antennocerebral tract in the MB calyx, presumably to maintain contact with the larval boutons (Fig. 7C2). Nearly all usp3 PPN axons exhibited grossly wild-type morphologies in adult LH; only one usp3 PPN axon in our sample failed to enter the LH. In summary, usp3 PPNs display ectopic processes in AL and MB that appear to be due to defects in pruning during early metamorphosis; these pruning defects do not seem to interfere with the growth or even targeting (in the case of AL) of adult-specific processes.
|
In summary, both usp3 and baboFd4 PPNs exhibit phenotypes in the adult brain consistent with blockage of pruning during early metamorphosis, including extraglomerular processes in the AL as well as large larval-like boutons on the main trunk and diversion from the inner antennocerebral tract as the axon passes through the MB calyx. However, baboFd4 PPNs also feature more severe phenotypes, particularly a complete lack of glomerular innervation and of adult-like axon collaterals with terminal swellings in the MB calyx, as well as failure to enter the LH and/or to elaborate higher order terminal branches. These latter phenotypes appear to be qualitatively different from those attributable to a simple loss of pruning, suggesting that TGFß signaling via baboon may have an additional role in re-extension and/or adult-specific targeting during metamorphosis.
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Discussion |
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PPNs function in both larval and adult olfactory circuits
We have shown that PPNs serve as relay interneurons connecting the antennal
lobe to the MB calyx and the presumptive LH in larvae, just as previously
characterized larval-born projection neurons do in adults. Each PPN generally
targets its dendrites to one glomerular substructure in the larval AL,
probably receiving input from one of the 21 olfactory receptor neurons of the
dorsal organ. From there, the PPN's axon extends to higher brain centers,
forming one or two large synaptic structures en passant on its way through the
MB calyx to the LH. Our electron microscopy studies with genetically encoded
markers expressed separately in PPNs or in MB neurons established that
PPNs form functional synapses in the larval circuit and that MB
neurons are among their postsynaptic partners.
Our analysis of these PPNs in the adult olfactory circuit confirmed and
extended the developmental and wiring logic derived from our previous analysis
of larval-born PNs. Just like larval-born PNs
(Jefferis et al., 2001),
embryonic-born PPNs are prespecified to target their dendrites to particular
glomeruli according to their birth order. Specifically, most PPNs are derived
from the same anterodorsal neuroblast that later gives rise to about half the
GH146-positive PNs. Like the larval-born PNs
(Marin et al., 2002
), PPNs
exhibit stereotyped terminal arborization patterns in the LH (see also
Wong et al., 2002
).
Interestingly, in the adult AL, PPNs innervate a distinct subset of glomeruli
from either their larval-born anterodorsal cousins or the projection neurons
generated by the lateral neuroblast. This indicates that, in addition to
relaying activity from larva-specific olfactory receptor neurons earlier in
development, PPNs expand the olfactory repertoire of the adult circuit.
As odorant response profiles of individual ORs are systematically mapped
(Dobritsa et al., 2003;
Hallem et al., 2004
), and
glomerular projection patterns of individual ORN classes are determined in
both the adult (e.g. Komiyama et al.,
2004
; Vosshall et al.,
2000
) and in the larval antennal lobe in the future, it will be
extremely interesting to compare whether these PPNs represent similar or
distinct odor repertoires at different life stages. At this point, one could
speculate that the PPNs represent odors that possess ethological significance
for both larvae and adult flies, perhaps by forming adult-specific connections
with ORNs expressing the same olfactory receptors found in larvae. However,
PPNs may be collectively no different from the larval-born PNs in the adult
olfactory system and may simply follow the same rules of connectivity
previously suggested, with PNs that innervate neighboring glomeruli in the AL
also targeting similar regions of the LH
(Marin et al., 2002
).
Implications for the development of the adult olfactory circuit
In addition to serving larval-specific functions, one proposed function for
larval circuits is to provide a foundation upon which adult circuits can be
built. In the case of the olfactory circuit, however, our previous analysis
indicated that the adult-specific antennal lobes form adjacent to, but
spatially distinct from, the larval antennal lobe
(Jefferis et al., 2004). Our
analysis of PPN remodeling supports the notion that the adult circuit is
constructed de novo rather than upon the larval circuit. A developmental
timecourse analysis revealed that PPNs prune their dendrites and axon branches
during early metamorphosis, so that only the main unbranched process from the
cell body to the distal edge of the calyx remains by 12 hours APF
(Fig. 3). By contrast, the
larval-born PNs begin to elaborate dendrites at the onset of puparium
formation and restrict their processes to specific regions of the developing
AL between 6 and 12 hours APF (Jefferis et
al., 2004
). Persistent projection neurons start exhibiting this
type of localized dendritic outgrowth in the adjacent but distinct adult AL
site only at 18 hours APF, around the time that adult-specific ORN axons
arrive but prior to their invasion of the AL. This strongly implies that, far
from providing contact-mediated cues for differentiating larval-born PNs, PPNs
target glomeruli in the developing AL only after the larval-born PNs have
established their dendritic target domains. The finding that PPN-specific
glomeruli are intercalated with those targeted by dendrites of larval-born
PNs, rather than occupying a spatially segregated domain in the adult antennal
lobe, implies complex targeting rules in the establishment of wiring
specificity of the adult circuit.
Relationship between pruning and re-extension
The fact that PPNs have clearly identifiable addresses for their dendritic
targeting in the adult circuit allowed us to ask the interesting question:
does assembly of the adult circuit depend on the disassembly of the larval
circuit? Our data suggest that neuronal reorganization appears to be separable
into two at least partially independent events, pruning and re-extension. Even
usp3 PPNs whose larva-specific dendrites and axons appear
unpruned still exhibit the random fine filopodial extensions characteristic of
wild-type neurons at 8-12 hours APF (data not shown), and moreover target
their new dendrites to appropriate adult antennal lobe glomeruli, as well as
exhibiting adult-specific axon collaterals in the MB calyx and grossly
wild-type terminal branches in the LH (Fig.
7).
The fact that most usp3 persistent PNs still innervate
appropriate glomeruli in the adult antennal lobe and have axons with adult
characteristics would suggest that ultraspiracle-mediated execution
of ecdysone signaling is required for pruning but not for responding to
re-extension and/or targeting cues in the developing brain. However, most
baboFd4 PPNs failed to target appropriately in the adult
olfactory system. This difference in phenotypes may be due to differential
perdurance of wild-type Usp versus Babo protein in single-cell MARCM clones
and/or to differences in the severity of the alleles examined, consistent with
the observation that baboFd4 PPNs show slightly more
homogeneous pruning phenotypes at 8 hours APF
(Fig. 5F). However,
usp3 carries a missense mutation that alters an invariant
arginine in the DNA-binding domain and blocks MB neuron pruning
completely (Lee et al., 2000
).
Thus, we favor the possibility that baboon is required for additional
ultraspiracle-independent functions during metamorphosis, in the
initiation of pruning, re-extension and/or targeting of adult olfactory
structures.
Regulation of neural circuit remodeling during metamorphosis
Since the original description of dendritic remodeling in an identified
Manduca motoneuron during metamorphosis
(Truman and Reiss, 1976),
neuronal remodeling during insect metamorphosis has been extensively studied,
in particular for motoneurons of Manduca and Drosophila
(reviewed by Levine et al.,
1995
; Tissot and Stocker,
2000
). Most of these studies have been conducted in individual
identified neurons. Our study of PPN remodeling reported here, in conjunction
with previous studies of MB
neurons that we have shown to be
postsynaptic targets of PPNs (Fig.
5), offers a unique opportunity to examine how synaptic partners
coordinate their remodeling.
We find that within each individual class of neurons, the timing of axon
and dendrite pruning are not necessarily synchronized. For instance, MB
neurons prune their dendrites (4-8 hours APF) prior to their
larval-specific axon branches (6-18 hours APF)
(Watts et al., 2003
).
Likewise, PPNs prune their dendrites (2-6 hours APF) slightly before axon
terminals in MB and LH (4-12 hours APF). However, pruning of PPN axon
terminals and MB
neuron dendrites in the calyx appears to be
coincident, raising the possibility that the pruning of synaptic partners
might be coordinated, or might even influence one another.
However, genetic studies indicate that there is at least some degree of
independence between these partner neurons. Given that remodeling occurs
during metamorphosis, regulation by the steroid hormone ecdysone had been
strongly implicated (Levine et al.,
1995). Earlier studies in Manduca using surgical and
endocrinological manipulations revealed that muscle degeneration and
motoneuron dendritic pruning and death are independently regulated by edcysone
(Weeks and Truman, 1985
).
Genetic tests for cell-autonomy had been performed using single-cell mutant
and cell-type-specific rescue analysis in MB neurons
(Lee et al., 1999
) and
peptidergic Tv neurons (Schubiger et al.,
2003
). In most other cases it has not been unequivocally
determined whether ecdysone acts directly on the remodeling neurons, or
whether some of the effects of ecdysone could be exerted indirectly through
the environment, including their synaptic partners. Single-cell clone analyses
using babo or usp mutants in this study strongly suggested
that ecdysone also acts directly on each individual PPN to regulate axon and
dendrite pruning. Thus, PPN axon pruning in MB calyx cannot be entirely a
consequence of pruning of its postsynaptic partners, just as MB
neuron
dendrite pruning cannot be entirely a consequence of pruning of its
presynaptic partners. Rather, they are developmentally programmed
independently, although, interestingly, they use the same mechanisms involving
TGF-ß induced expression of EcR-B1, which works together with its
co-receptor Usp to regulate gene expression.
These experiments by no means exclude the possibility that PN axon pruning
and MB neuron dendrite pruning are to some degree interdependent. For
instance, it could be that ecdysone signaling confers competence to prune on
each class of neurons, but the execution of the pruning process would depend
on further cellular interactions, including those between synaptic partners.
Future genetic manipulations that block pruning of one class of neurons and
examine the consequences for the other class will shed light on the
contribution of cellular interactions in neural circuit remodeling.
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ACKNOWLEDGMENTS |
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