1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
2 Neurosciences Program, Stanford University, Stanford, CA 94305, USA
3 Department of Biology, University of Fribourg, CH-1700 Fribourg,
Switzerland
4 Institute of Molecular and Cellular Biosciences, University of Tokyo,
Japan
5 The Graduate University for Advanced Studies, Japan
Author for correspondence (e-mail:
lluo{at}stanford.edu)
Accepted 1 October 2003
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SUMMARY |
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Key words: Dendrites, Dendritic development, Antennal lobe, Projection neurons, Drosophila, Olfaction
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Introduction |
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The developmental origins of this punctate olfactory map remain somewhat
mysterious. Experiments in mice have demonstrated an apparent role for the
olfactory receptors in this process (Wang
et al., 1998; Vassalli et al.,
2002
) but showed no substantial role for activity in the
establishment of the map (Lin et al.,
2000
; Zheng et al.,
2000
). Recent studies in Drosophila
(Gao et al., 2000
;
Vosshall et al., 2000
) have
highlighted the organizational similarities of vertebrate and insect olfactory
systems (reviewed in Strausfeld and
Hildebrand, 1999
), opening up another model system to address the
developmental origins of the olfactory map.
The antennal lobe (AL) is the first olfactory centre in the
Drosophila brain (Fig.
1A). Structurally it is a flattened sphere of maximum diameter
80 µm subdivided into about 50 glomeruli
(Laissue et al., 1999
;
Kondoh et al., 2003
), which
are the functional units of the olfactory map. In cellular terms, it consists
of the cytoplasmic processes (but not cell bodies) of four major cell types:
the axonal terminals of olfactory receptor neurons (ORNs) and the dendrites of
second-order projection neurons (PNs) (both of which usually invade single
glomeruli), local interneurons (LNs) [whose processes ramify through large
areas of the lobe, synapsing in many glomeruli
(Stocker et al., 1990
)] and,
finally, glial processes ensheathing the glomeruli
(Jhaveri et al., 2000
).
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Here, we present a systematic study of the development of PNs down to single-cell resolution. We find that specific connectivity in the AL develops in three phases. First, PN dendrites show active targeting to specific regions of the developing AL before the arrival of their partner ORN axons; a prototypic map resembling the adult AL therefore forms before any physical contact with ORN axons. Second, glomerular development proceeds rapidly after the arrival of ORNs and a largely mature glomerular organisation is reached before significant glial ingrowth. Finally, glomeruli increase uniformly in size and are wrapped by glia. These studies reveal two previously unexpected findings, which might be of general significance in neural development. First, PN dendrites are capable of autonomous growth and pattern formation in the absence of their future presynaptic partners. Second, our analysis does not support the existence of a third-party guidepost cells or processes within the developing AL that serve as origins of pattern formation; instead, we propose that the olfactory map originates both from patterning information external to the developing AL and from interactions among PN dendrites.
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Materials and methods |
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Immunochemistry
Fixation, immunochemistry and imaging were carried out as described
(Jefferis et al., 2001).
Additional antibodies used in this study were: the mouse monoclonal 9F8A9 and
the rat monoclonal 7E8A10 against Elav, both at1:20 dilution; the mouse
monoclonal 8D12 against Repo at 1:10 dilution (all DSHB, University of Iowa);
rat monoclonal anti-N-cadherin extracellular domain, at 1:40 dilution (T.
Uemura, Kyoto University); rabbit polyclonal antibody against green
fluorescent protein (GFP), 1:800 (Molecular Probes); Cy5-conjugated goat
anti-rat/mouse IgG at 1:400 dilution (Jackson). General nuclear staining was
with TOTO-3 for 30 minutes at 1:1000 of the stock concentration (Molecular
Probes).
Defining the border of the developing AL
N-Cadherin staining was used as the reference for defining the extent of
the developing AL. When N-cadherin was not used, anti-fasciclin-II (FasII)
antibody and/or TOTO-3 staining (for nuclei) were used to trace the outline of
the lobe. In the case of TOTO-3, this generated a clear if slightly less
accurate border. In the case of FasII, three axon tract branch points were
used as fiducial points in conjunction with weak FasII staining in the core of
the developing AL; this allowed the lobe outline to be estimated reliably.
This approach was validated by blind labelling of the AL outline using the
FasII channel in confocal stacks of brains triple labelled for GH146,
N-cadherin and FasII.
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Results |
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Timing of ORN axon development
Adult-specific ORNs (hereafter referred to simply as ORNs) are born during
early pupal development and send their axons towards the AL shortly
thereafter. Two previous studies (Tissot
et al., 1997; Jhaveri et al.,
2000
) have described the timing of ORN axon arrival at the
developing AL using several enhancer-trap lines expressed in subsets of ORNs.
Jhaveri et al. (Jhaveri et al.,
2000
) observed that pioneering ORN axons begin to contact the lobe
18 hours after puparium formation (APF) and start to invade the lobe 20 hours
APF. Besides confirming that developmental staging was consistent between
laboratories, we felt that a re-examination of the time of ORN axon arrival
was important for two reasons. First, we have found that N-cadherin labels the
developing AL (H. Zhu, personal communication), providing an objective
criterion to define this structure that was not available in earlier studies.
Second, we examined contributions from all ORNs to exclude the possibility
that some particularly precocious ORNs would not be labelled by the enhancer
trap lines used in earlier studies.
In order to label ORN axons of all types, we used GAL4-C155, an
enhancer trap of the elav locus expressed in all post-mitotic neurons
as soon as they are born (Robinow and
White, 1988). However, because we did not wish to label neurons
within the brain, we combined this with the MARCM strategy
(Lee and Luo, 1999
) and the
eyeless-FLP element (Newsome et
al., 2000
); in addition to the eye disc, ey-FLP drives
mitotic recombination in the antennal disc and maxillary palp where ORN cell
bodies reside (Hummel et al.,
2003
) (Fig. 2A).
This strategy results in random labelling of up to half of the ORNs in any
particular antennal disc (Fig.
2B2). Because GAL4-C155 shows pan-neural
expression and clone generation is random, by examining several samples we can
be assured of studying all classes of ORN axons.
We found at 14 hours APF that there is already a thin axon bundle exiting
the third antennal segment, where ORN cell bodies are located. Over the next 4
hours, the axon bundle becomes much thicker as more ORNs differentiate (data
not shown). When do these axons reach their target? At 14 hours APF, ORN axons
(Fig. 2C-H green) have not yet
contacted the developing AL (outlined by N-cadherin,
Fig. 2C-E, red) (six
hemispheres examined). By 16 hours APF, they have just reached the AL in most
cases examined (10/14 hemispheres) and, at 18 hours APF, we observe contact of
ORN axons with the ventral edge of the developing AL in all samples examined
(12/12). This finding is consistent with those of Jhaveri et al.
(Jhaveri et al., 2000). At 18
hours APF, ORN axons circumscribe the developing AL extending towards the
midline; by 20 hours APF, axons reach the midline as they project to the
contralateral AL. Significant ORN axon invasion of the AL is evident at 26
hours APF (Fig. 2F-H). As late
as 26 hours APF, there is no evidence for the arrival of maxillary palp ORN
axons, which should be distinguishable from antennal ORNs because they enter
the lobe from a more ventral location. In summary, ORN axons reach the
periphery of the lobe 16-18 hours APF and do not invade the lobe until 24-26
hours APF.
PNs extend axons and then dendrites
The enhancer trap GAL4-GH146
(Stocker et al., 1997) labels
90 of the estimated 150 PNs; the lineage
(Jefferis et al., 2001
), and
mature organization (Marin et al.,
2002
; Wong et al.,
2002
) of GH146-positive PNs have been extensively
characterized. To study a subset of these GH146 PNs, we used the
MARCM system (Lee and Luo,
1999
) to generate labelled clones of two types: single cells
(products of terminal division after heat-shock-induced mitotic recombination)
and neuroblast clones (containing all the remaining cells within a neuroblast
lineage after the time of heat shock). Earlier studies have identified two
major PN lineages, each derived from a single neuroblast, and 2 corresponding
groups of neurons with cell bodies anterodorsal and lateral to the mature AL
(adPNs and lPNs, respectively) (Jefferis
et al., 2001
). Extensive studies of the adPN lineage demonstrated
that different classes of PNs are born sequentially; for example, single cell
clones generated within the first 24 hours of larval hatching always send
their dendrites to the glomerulus DL1. This observation is of significant
technical value because we can follow the development of these DL1 PNs long
before any glomeruli are evident. In addition, it should be emphasized that
neuroblast clones generated at this stage or later are exclusively composed of
adult-specific PNs.
After their birth, these adult-specific PNs extend axons towards higher olfactory centres in the larval brain. At 72 hours after larval hatching, axons do not seem to have reached these structures but, by the wandering third instar stage, axons have extended to the mushroom body calyx and a presumptive lateral horn area (data not shown). At this stage, PNs have no dendritic or axonal terminal arborizations. In short, although adult-specific PNs might use the existing axonal tracts of persistent PNs for axon guidance, they are not integrated into the larval olfactory system.
The first signs of dendritic growth can be observed at the wandering third
instar stage (Fig. 3A) and
growth is evident by the time of puparium formation
(Fig. 3B). At this point,
dendrites appear as a swelling, grow radially outwards from the axonal bundle
and lack any obvious organisation. Intriguingly, this growth is at a novel
site outside the existing larval AL: there is a lack of overlap between the
synaptic marker nc82, which stains the larval AL
(Fig. 3B, dotted outline), and
the new dendritic growth (Fig.
3B, arrowheads). In short, it appears that PN dendrites are
generating a new structure rather than invading an existing one. By comparing
single cell and neuroblast clones at this time, it appears that the single
cell dendrites are diffuse but might extend through a large fraction of the
volume occupied by an 30 cell neuroblast clone
(Fig.
3B1,B3). This observation applies both to
DL1 PNs (Fig. 3B3)
and to other classes of PNs labelled by later heat shocks (data not shown).
Thus, at this stage, dendrites are not restricted into separate regions.
PN dendrites rapidly restrict into discrete areas of the developing AL
By 6 hours APF, there has been significant dendritic growth, so that the
volume occupied by adPN dendrites is now similar to that occupied by the
larval lobe (Fig.
3C1). However, inspection of single cells does not show
a large increase in the volume through which their dendrites extend
(Fig.
3B3,C3). A possible explanation is that the
dendrites of individual neurons are starting to sort into individual regions,
reducing their degree of overlap; a group of neurons would therefore occupy
more space even though individual dendritic arbours might not change in
extent. Evidence for this is provided by the appearance of lPNs at this time
(6 hours APF) (Fig.
3C2). These are less homogeneous than adPN dendrites,
showing discrete areas of extension and other uninnervated areas. This
impression is strengthened by examining lPN dendrites at 12 hours APF
(Fig. 3D2).
Dendrites accumulate only at certain regions of the developing lobe, whereas
large areas are almost devoid of lPN dendrites (e.g. asterisk in
Fig. 3D2). In fact,
it can be seen that this central unoccupied area in
Fig. 3D2 corresponds
roughly with the zone of innervation of adNb dendrites in
Fig. 3D1. These
reproducible patterns of innervation suggest that the dendrites of these two
different groups already only innervate specific regions of the lobe.
Significantly, this is not just because dendritic growth is restricted to a
region immediately adjacent to the cell bodies of these two classes of
projection neurons the arrowhead in
Fig. 3D2 indicates a
group of dendrites some distance from the lPN cell bodies. These observations
suggest active targeting of dendrites to regions of the developing AL.
Dendritic patterning before ORN axon arrival
From the data presented so far, it is clear that, by 12 hours APF, PN
dendrites occupy restricted regions of the developing lobe. Furthermore,the
gross innervation patterns of adPNs and lPNs, two specific sets of 30
neurons, show signs of localizing to specific regions of the lobe. This
touches on a key question: do the dendrites of any particular neuron occupy a
restricted, specific portion of the lobe?
Fig. 4 addresses this question
at 18 hours APF, up to which point PN development is independent of ORN axons
(Fig. 2).
As described earlier, MARCM clones generated within the first 24 hours after larval hatching always innervate the glomerulus DL1. We can therefore test whether, at this early stage in development, when no glomeruli can be discerned, these DL1 PNs target a specific region of the developing lobe.
Fig. 4A shows a single
optical section through the middle of a pair of ALs in a rare brain with DL1
single cell clones in both hemispheres. The margin of the lobe can be traced
out by the border of cell nuclei stained with TOTO-3 (blue); orientation is
also provided by staining for the FasII marker, which dynamically stains axon
tracts during the first third of pupal development along with some weak
staining of processes within the AL. It can be seen that the dendritic arbours
of both DL1 neurons are specifically localized in the dorsolateral corner of
the lobe this is consistent with the eventual position of DL1
(Laissue et al., 1999)
(Fig. 3J3). We
analysed the position of 20 such single cell clones and found that this
location was consistent among all samples examined.
DL1 dendrites occupy a specific portion of the dorsal AL. However, this is relatively close to their cell bodies, so there is one trivial explanation that must be excluded that these dendrites are localized because they have not yet been able to grow further into the AL. For comparison, therefore, a pair of anterodorsal neuroblast clones is presented in Fig. 4B, imaged at the same depth in the AL. The dendrites of these two neuroblast clones extend through a large area at this level of the lobe. This indicates that the DL1 dendrites in Fig. 4A are not just in a specific part of the AL because of a very restricted distribution of all adPN dendrites; DL1 dendrites actively target a distinct region of the AL compared with other neurons of this lineage.
Do all individual projection neurons behave in the same way as DL1 PNs?
This is technically difficult to answer with precision, because DL1 PNs are
the only class that we can unambiguously identify by their birth-date.
Nevertheless, we generated a distinct set of clones by heat-shocking larvae at
72 hours after larval hatching and dissecting them at 18 hours APF; none of
these clones will be DL1 PNs (Jefferis et
al., 2001). All clones had spatially restricted dendrites in
different parts of the AL, as exemplified by
Fig. 4F from a brain in which
two single cells were labelled. In fact, the other 27 single cell clones that
we generated could be classified into three main classes, according to their
dendritic location. 19/27 cells (Fig.
4C, Class I) had dendrites on the lateral aspect of the lobe; 5/27
cells (Fig. 4D, Class II)
innervated the dorsomedial corner of the AL; 3/27 cells
(Fig. 4E, Class III) actually
had two branches. These observations are consistent with the notion that
individual PNs occupy a restricted, specific portion of the AL. This is
further supported by the mirror symmetry of neuroblast clones that occupy
specific regions of the AL (e.g. Fig.
4B, arrowheads).
Maturation of the glomerular map after ORN axon invasion
From 18-40 hours APF, there is a progressive refinement of the projection
pattern of PN dendrites, although no glomeruli can be detected by the nc82
synaptic marker. In fact, even in neuroblast clones at 18 hours APF, tentative
assignments can even be made for some dendritic foci for example, the
arrowheads in Fig. 4B are
probably an accumulation of dendrites from VA1d and VA1lm PNs. Similarly,
glomerulus VA3, which is a ventral glomerulus well separated from other adPN
glomeruli (e.g. arrow in Fig.
3I1), is the probable target of the ventromedial
accumulation of dendrites that can be seen in the left hemisphere in
Fig. 4B (arrow). By 40 hours
APF, nearly all dendritic accumulations can be identified in confocal stacks
(compare, for example, Fig.
3H1 with Fig.
3J1).
During the first 18 hours of pupal development, the intensity of nc82 staining diminishes rapidly so that, by 18 hours, it is no longer detectable; this reflects the degeneration of the larval AL (see below). At 30 hours and 40 hours, there are signs of a reappearance of specific nc82 staining; however, distinct glomeruli are only detectable using this marker by 50 hours APF. At this time, the dendritic organization is essentially mature. In Fig. 3I1, for example, all of the major adNb glomeruli can be matched to their adult counterparts in Fig. 3J1; in this z projection, however, it is hard to get an idea of how well contained dendrites are within a single glomerulus. Fig. 3I2 is a single optical section through a lateral neuroblast clone. All four glomeruli (DA1, VA5, VA7m, DA2 clockwise from top right) are clearly identifiable and innervated without any obvious dendritic spill-over to neighbouring glomeruli. These results are corroborated by the appearance of single cell DL1 clones, which also have a compact dendritic tree restricted to a single glomerulus that can be identified by nc82 staining alone.
The major change from 50 hours APF to adult appears to be a large increase in the size of the AL. The increase is about 50% in linear dimensions or fourfold in volume. A large proportion of this increase results from local increases in the density of existing neuronal processes, with additional contribution from ingrowth of glial processes (see later).
Following PN development using a subtype-specific PN marker
Our initial studies make use of GH146, which labels a large subset of PNs.
Although we were able to focus on small numbers of GH146-positive neurons
using the MARCM system, we sought to complement these studies by identifying
enhancer trap lines labelling specific subsets of PNs. After testing a panel
of GAL4 enhancer trap lines (Hayashi et
al., 2002), we chose to focus particularly on the line
GAL4-Mz19; in adults, it specifically labels PNs that innervate two
large glomeruli, VA1d and DA1, on the anterior surface of the AL, along with a
less distinct and more posterior glomerulus DC3
(Fig. 5F). We can then ask when
specific innervation of these glomeruli can be observed during
development.
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Development of axon terminal arborizations
In the adult fly, PNs of the same glomerular class have stereotyped axon
terminal arborization patterns in a higher olfactory centre, the lateral horn
(Marin et al., 2002;
Wong et al., 2002
). What is
the relative timing of the development of specific dendritic targeting and
specific axonal arborizations? Fig.
6 presents a time course of axonal development for single cell DL1
clones. At early pupal stages, axonal development appears to lag somewhat: at
0 hours and 6 hours APF, there is no sign of terminal extension in the
mushroom body calyx or lateral horn. By 12 hours APF, however, there are signs
of sprouting in these regions (Fig.
6C, blue and red asterisks). At 18 hours APF in the mushroom body,
this early sprouting has resolved itself into several distinct collaterals
(Fig. 6D, blue arrowhead). In
the lateral horn, there is further extension of the main axon branch but no
extension of the dorsal collateral, which is very characteristic of DL1
projection neurons (compare Fig. 6D and
6I). At 24 hours APF, however, there is the first sign of this
dorsal collateral at this point, a stump of about 5 µm
(Fig. 6E, red arrowhead). By 30
hours APF, this has become more substantial about 15 µm in the
example shown. It was possible to discern a clear collateral in 8/10 samples
at this stage (Marin et al.,
2002
). In the 40 hours APF sample, there is actually a second
dorsal collateral present; this is sometimes observed in pupal stages but
never persists in the adult. There is also substantially enhanced terminal
sprouting (Fig. 6G, green
arrowhead). By 50 hours APF, the axon projection pattern is quite mature
mushroom body collaterals have formed a terminal ball, as observed in
the adult, and the lateral horn dorsal collateral has reached almost its full
extent. However, the terminal arborizations of the main branch in the lateral
horn have not yet obtained their final form. This time point is significant
because it is just before the earliest known expression of an olfactory
receptor in Drosophila (Clyne et
al., 1999
) and before mature synapses have been detected in the
lobe (Devaud et al., 2003
).
Patterned sensory activity is therefore unlikely to play an instructive role
in development of either PN dendritic targeting or, at least for DL1 PNs, most
aspects of axon terminal arborisation.
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We determined the time of degeneration of the larval lobe using nc82, a presumed synaptic marker, and N-cadherin, a marker enriched in the developing adult-specific lobe. In addition, we used GH146 directly, without generating MARCM clones, so both persistent and adult-specific PNs are labelled. At 0 hours APF (Fig. 7A), the larval lobe (dotted outline) is strongly stained by nc82 and also by N-cadherin; this is also where most GH146-positive dendrites are concentrated (Fig. 7A2). However, there is a second area (solid outline) dorsal and posterior to the larval lobe with a lower concentration of GH146-positive dendrites, higher levels of N-cadherin and no nc82. This area matches in position the location of newly developing dendrites described in the neuroblast and single cell clones in Fig. 3B. Thus, at this stage the intact larval lobe and newly developing adult lobes are clearly distinct.
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By 12 hours APF, nc82 staining is very much reduced (Fig. 7C4) and the larval lobe is no longer clearly demarcated, although there is an area of nc82 and weaker N-cadherin staining along the ventrolateral margin of the lobe (dotted lines in Fig. 7C). There are some GH146-positive dendrites within this marginal zone that are continuous with those in the rest of lobe (Fig. 7C1-4, arrowhead), suggesting that the two structures might be effectively fused at this stage. This is consistent with the results in Fig. 3D, in which the dendrites of adPN and lPN clones showed some overlap with the remaining nc82-positive larval area. In summary, the larval lobe is intact at 0 hours APF, whereas an adjacent adult structure, posterior and dorsal to the larval lobe, starts to show dendritic elaboration; by 6 hours APF, larval dendrites have degenerated significantly whereas adult-specific dendrites have grown significantly; at 12 hours the adult lobe is largely continuous with what remains of the larval structure.
What is the status of the larval ORNs during this time? We examined this
question using the enhancer trap line GAL4-MJ94
(Joiner and Griffith, 1999),
which labels all the receptor neurons of the larval olfactory and gustatory
organs (R.F.S., unpublished); similar results (data not shown) were obtained
with a second line GAL4-JFF-4551, which labels two-thirds of larval
ORNs (Python and Stocker,
2002
). At 0 hours APF, larval ORNs densely innervate the
nc82-positive larval lobe (dotted outline in
Fig. 7D). MJ94 driven CD8-GFP
can also be seen in the ORN axons in the antennal nerve, indicated by an
asterisk as it reaches the lobe. At 6 hours APF
(Fig. 7E), larval ORNs extend
through a similar area but now showing fingers of innervation (which are
coincident with weaker nc82 staining) separated by uninnervated areas. The
zone of larval innervation is largely distinct from the N-cadherin-positive
adult-specific lobe but there is some overlap along the anterior face of the
ventral part of the new lobe. By 12 hours APF, ORN axons have mostly pruned
back to a stump in the ventrolateral corner of the lobe. There are still,
however, about 10-15 neurites, which are bare except for occasional blebs of
up to 3 µm in diameter. Although these neurites extend in a few instances
up to 20 µm, their position on the anterior surface of, rather than within,
the adult-specific lobe indicates that they have limited contact with
adult-specific structures.
In conclusion, the larval lobe degenerates over the period from 0-12 hours APF; growth of the developing adult lobe occurs at a site adjacent to but distinct from this degenerating structure as long as the larval lobe remains identifiable. The spatial information allowing early dendritic patterning within the adult structure must therefore derive from cellular constituents other than remnants of the larval olfactory system.
Cellular cues for PN dendritic development constituents of the developing adult-specific lobe
We next investigated whether glia could provide positional cues for PN
dendritic targeting using an enhancer trap line GAL4-M1B, in which
GAL4 is under the control of the repo promoter and therefore
expressed in all central nervous system glia
(Xiong et al., 1994). We
confirmed that, near the developing AL, all nuclei are either Elav positive (a
pan-neural nuclear marker) or Repo positive (e.g.
Fig. 8B5), and
therefore all non-neuronal cells are Repo-positive glia. In addition, all
Repo-positive nuclei are surrounded by strong membrane staining when
GAL4-M1B is crossed to UAS-mCD8-GFP, a membrane marker;
however, there is some additional weak background staining in most other cells
in the brain. Therefore, mCD8-GFP driven from GAL4-M1B
should strongly label all glial processes. As expected, in adults, glial
processes can be seen wrapping individual glomeruli
(Fig. 8A5). Are
these processes present in the developing AL during the early phase of PN
dendritic targeting?
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Despite the lack of glial processes within the developing adult-specific
lobe, there are glial processes outside the lobe
(Fig. 8A) and double labelling
of GAL4-M1B-driven mCD8-GFP and anti-Repo antibody confirms
that, during the period 0-18 hours APF, there are scattered Repo-positive
nuclei surrounding the developing AL (Fig.
8B1-3). Glial processes could therefore contribute to
PN dendritic patterning, but only from outside the AL. In contrast to the lack
of glial processes, we found that mCD8-GFP driven by the pan-neural
GAL4-C155 fills the developing AL between 0 hours and 18 hours APF
(Fig. 8C). Because we have
ruled out a significant contribution by larval ORNs
(Fig. 7) and adult ORNs are yet
to enter the lobe (Fig. 2), the
remaining possibilities are LNs and PNs themselves. We could not conclusively
determine the contribution of LNs owing to lack of appropriate reagents.
Neither of the two LN GAL4 lines we have examined (GAL4-189Y and
GAD1-GAL4) drives marker expression in the adult-specific AL between
0 hours and 12 hours APF, although both label the larval lobe (data not
shown). However, GAL4-189Y only labels a few larval LNs
(Python and Stocker, 2002) and
GAD1-GAL4, which is derived from the promoter of the glutamic acid
decarboxylase gene and should therefore label all GABAergic inhibitory neurons
(Ng et al., 2002
), might not
turn on during early stages of LN development. Nevertheless, our inability to
find LN processes in the developing adult-specific AL in early stages of pupal
development is consistent with previous observations in moth that LN dendrites
invade protoglomeruli later than PNs
(Oland et al., 1990
).
Thus, although we cannot rule out a contribution of LNs, the evidence
accumulated so far suggests that PN dendrites are a major constituent of the
developing AL. Indeed, when comparing GAL4-C155 and
GAL4-GH146 (labelling 90 of an estimated 150 total PNs), in
anterior sections GH146-positive PN processes appear to occupy most of the
space labelled by GAL4-C155 (compare
Fig. 8B4 with
8C4,5). We have argued that structures internal to the AL
participate in patterning PN dendrites, and so we propose that PN-PN
interactions contribute significantly to the initial patterning of the
developing AL.
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Discussion |
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Dendritic growth and patterning independent of presynaptic axons
A key finding in this study is that PN dendritic development is
surprisingly independent of presynaptic ORN axons. It is generally thought
that dendritic growth and maturation are coupled with presynaptic axon
invasion and synapse formation, which might be significantly shaped by
electrical activity (reviewed by Cline,
2001). We found that, before ORN axons reach the developing AL, PN
dendrites have undergone significant growth and branching. More strikingly,
dendrites of different PN classes have created a pattern in the AL at 18 hours
APF that resembles the adult glomerular map, in the complete absence of their
presynaptic partners (Fig. 4).
This observation is in contrast to the prevailing view of olfactory
development (see below) and underlines a very significant dendritic
contribution to the origins of wiring specificity.
It is important to realize that this finding is not a natural prediction of
our previous findings that PNs are prespecified to synapse with a specific
class of ORNs by lineage and birth order
(Jefferis et al., 2001). For
instance, all three models depicted in Fig.
1B are consistent with the prespecification data. Our current
study clearly favours the second model, that PN dendrites are patterned first.
It is probable that gradual invasion of ORN axons from 24 hours APF
(Fig. 2) refines and
consolidates this prototypic dendritic map (see below), allowing rapid
development of the AL to essentially adult form by 50 hours APF.
Site selection of one synaptic partner before the other has also been
described recently in Caenorhabditis elegans
(Shen and Bargmann, 2003).
Interestingly, in that case, the presynaptic partner selects the synaptic site
in the absence of its postsynaptic partner by interacting with a third party
guidepost cell.
Origin of patterning information
What positional cues allow dendritic patterning before ORN axon arrival?
The first leading candidate to provide positional cues is the larval lobe
because it is presumably patterned for larval olfaction, albeit in a form
simpler than that of the adult AL (Tissot
et al., 1997; Python and
Stocker, 2002
). However, we find that, at early pupal stages, the
developing adult AL is clearly distinct from the larval lobe and is minimally
invaded by remnants of the degenerating larval lobe. Glia are a second
candidate and, indeed, glia have been suggested to play important roles in
sorting ORN axons into individual classes in the moth (Rossler et al., 1999)
and are the leading candidates in the mouse olfactory bulb to provide
positional cues for ORN axon targeting
(Bulfone et al., 1998
).
However, we did not find significant glial processes within the developing AL
between 0 hours and 18 hours APF, the critical period for PN dendritic
patterning. Although it is likely that glial cells and processes surrounding
the AL contribute to PN dendritic patterning, it is difficult to imagine that
the surrounding tissues could contribute all the positional cues that allow
dendrites of a specific class to occupy specific regions of the AL, which is a
three-dimensional sphere.
These analyses lead us to speculate that PN dendritedendrite interaction
might contribute significantly to the eventual patterning of PN dendrites for
the following reasons. First, PN dendrites appear to be a major constituent of
the developing adult-specific AL, which is composed predominantly, if not
exclusively, of neuronal processes (Fig.
8). Indeed, preliminary electron microscopic analysis using
genetically encoded electron microscopy markers suggests that the developing
AL is composed entirely of neuronal profiles and that GH146-positive PN
dendritic profiles are clustered together without intermingling with other
neuronal profiles (R. Watts and L.L., unpublished). Second, different classes
of PNs are probably endowed with molecular differences because of their
lineage and birth order (Jefferis et al.,
2001; Komiyama et al.,
2003
), which could be used to create heterogeneities in the
developing lobe. LN processes do remain a possible source of information that
we could neither find positive evidence for nor rule out. However, it is more
difficult to imagine how heterogeneity is created by LNs, because processes
from each LN occupy a large proportion of the AL, rather than individual
glomeruli (Stocker et al.,
1990
).
Based on these considerations, how do we envisage that PN patterning could occur in early pupa before ORN arrival? We propose a hierarchy of positional cues. Global cues, which could be diffusible or contact-mediated guidance molecules from outside the lobe, could allow dendrites to target to an approximate region of the developing lobe with respect to the body axes, causing, for example, glomerulus V to form ventrally and glomerulus D to form dorsally. Local dendritedendrite interactions would then allow PN dendrites of the same class to adhere tightly; dendrites destined to occupy neighbouring glomeruli would associate more weakly and/or be repelled. This hierarchy of cues seems to be essential for generating a structure that has global order and highly reproducible local spatial relationships among neighbouring PN classes. Our observations at 18 hours APF (Fig. 4) suggest that, although the global targeting of dendrites to specific regions of the AL is largely complete, the sorting process is not, because dendrites from neighbouring classes can still exhibit significant overlap. Continuing dendrite-dendrite interaction after 18 hours APF and interaction with ORN axons (see below) would further refine this prototypic dendritic map.
It is noteworthy that axon-axon interaction has already been shown to
contribute to the specificity of connections between Drosophila
photoreceptors and their targets (Clandinin
and Zipursky, 2000). Although we are not aware of a proposal for
such mutual dendrite sorting, the molecular mechanisms could be related to
dendrite tiling (Wassle et al.,
1981
; Grueber et al.,
2002
; Grueber et al.,
2003
). In tiling, dendrites of the same neuronal class show
homotypic repulsive interactions; we would propose that homotypic attraction
and/or heterotypic repulsion from neighbouring dendritic classes could
contribute to dendritic sorting in the developing AL.
Contributions of ORNs to the developing olfactory system
The observation that in Drosophila PN patterning precedes that of
ORNs seems at odds with existing observations in insects and vertebrates,
emphasizing the primary role of ORN axons in organising glomerular development
(e.g. Oland et al., 1990;
Valverde et al., 1992
;
Malun and Brunjes, 1996
;
Treloar et al., 1999
). In all
these studies, glomerular formation was first evident in the accumulation of
ORN axons in protoglomeruli. This organizational function of ORNs is further
supported by several perturbation experiments. For instance, in the moth
Manduca sexta, developmental deantennation prevents normal glomerular
formation (Boeckh and Tolbert,
1993
), whereas surgical removal of a subset of PNs still permits
ORNs to form relatively normal glomerular terminations outlined by glial cells
(Oland and Tolbert, 1998
).
Similarly, genetic ablation of most mitral or granule cells in mutant mice
still permits axonal convergence of specific ORN classes, although the
structure of the olfactory bulb is disorganized
(Bulfone et al., 1998
). In
Drosophila, it was reported that in atonal mutants,
formation of a proposed pioneer class of ORNs is disrupted, axonal targeting
of other ORNs is delayed and glial processes are disturbed. It was further
suggested that PN development must depend on ORNs because, in atonal
mutants, PN patterning [as visualized by GH146 staining (labelling
90
PNs)] is disrupted (Jhaveri and Rodrigues,
2002
).
Many of these apparent contradictions can be resolved by treating spatial
patterning and glomerular formation as two distinct processes. An important
technical improvement of our study is that we are able to visualize the
dendritic fields of identifiable PNs down to the single cell level with MARCM
or small groups of cells with GAL4-Mz19. We can therefore describe
much earlier developmental events with higher anatomical resolution,
demonstrating the existence of spatial patterning well before glomeruli are
morphologically distinct and, indeed, before one major component of the
glomeruli the ORN axons is even present. For example, in
Fig. 4A, we can see that
dendrites of DL1 PNs occupy a discrete and specific location in the developing
lobe. However, because their dendrites still overlap with other PN classes,
this patterning is not that obvious when looking at larger numbers of neurons
(e.g. Fig. 4B) and is scarcely
apparent at all when more cells are visualized
(Fig. 8B4). Indeed,
some existing single cell labelling studies have shown that PNs in
Manduca have restricted dendritic arborizations before any glomerular
patterning is evident (Malun et al.,
1994). However, without being able to label specific classes of
PNs, such studies cannot determine whether PN dendrites are in a spatially
appropriate location.
How do ORN axons come into register with the prototypic PN map when they
reach the lobe? At one extreme, ORN patterning might be completely dependent
on PN patterning. ORN axons could simply recognize specific classes of
pre-patterned PN dendrites through receptor-ligand or homophilic interactions.
Alternatively, PN patterning could generate a third-party map, which is
recognized by ORNs. Although certainly consistent with the developmental
studies described here, these models are not supported by experiments in other
organisms such as the target neuron ablation experiments of Oland and Tolbert
(Oland and Tolbert, 1998) in
moth and Bulfone et al. (Bulfone et al.,
1998
) in mice. In addition, transplantation experiments in moth
(Rossler et al., 1999) or the formation of a novel glomerulus upon expression
of a rat olfactory receptor in mice
(Belluscio et al., 2002
),
indicate that ORNs might have substantial autonomous patterning ability. At
the other extreme, patterning of ORN axons could be completely independent of
the prototypic map created by PN dendrites. In theory, ORN axons could
recognize third-party cues previously used to pattern PN dendrites. However,
we are unable to find such third-party cues within the developing AL. Of
course, ORNs could still be patterned by PN independent cues external to the
lobe and axon-axon interactions, in a manner directly analogous to our
hypothesis for PN patterning. In the case of such strict independence, ORNs
expressing a particular receptor would form specific connections with partner
PNs, rather than inappropriate adjacent PNs, only because both sets of neurons
target with great precision to exactly the same spatial location. This degree
of independence seems both implausible and inefficient.
Instead, we propose that both ORN axons and PN dendrites have substantial autonomous patterning ability for instance through PN-PN or ORN-ORN mutual interactions and interactions with cellular cues surrounding the developing AL but that the two resultant proto-maps interact during development to generate the final mature glomerular organisation. This proposal is the most parsimonious explanation for the existing data supporting the organizational functions of ORNs as summarized above, our data described in this study and our preliminary observations that ORN axon targeting appears to be resistant to PN perturbation (T. Chihara, H. Zhu and L.L., unpublished). It also has the advantage of robustness each map could reinforce and refine the other, resulting in a precise match between pre- and postsynaptic partners without the necessity for extreme targeting precision. Furthermore, although two maps might seem to be more complicated than one, they need not be molecularly more complex. Many of the molecules used for PN patterning could also be used for ORN patterning or for interactions between ORNs and PNs. Having described with great precision the cellular and developmental events that lead to the patterning of the AL, we are now in a good position to attack the molecular basis of wiring specificity in the Drosophila olfactory system.
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ACKNOWLEDGMENTS |
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Footnotes |
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