1 Department of Molecular and Integrative Physiology, University of Illinois,
Urbana, IL 61801, USA
2 Department of Life Science, National Tsing Hua University, Hsinchu, 30043,
Taiwan
3 Department of Cell and Structural Biology, University of Illinois, Urbana, IL
61801, USA
* Author for correspondence (e-mail: tzumin{at}life.uiuc.edu)
Accepted 4 March 2003
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SUMMARY |
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Key words: Drosophila, Mushroom body, Dendritic elaboration, Mosaic analysis, Remodeling
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INTRODUCTION |
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One Drosophila MB is derived from four neuroblasts (Nbs)
(Ito et al., 1997), each of
which sequentially generates three distinct types of MB neurons that can be
distinguished based on their axon projection/fasciculation patterns
(Lee et al., 1999
). The cell
bodies of MB neurons are clustered on the dorsal posterior surface of the
protocerebrum. Each cell body sends out one primary neurite that gives rise to
dendritic branches and then extends ventrally and anteriorly through the
peduncle (e.g. Fig. 4A-C). The
peduncle ends near to the anterior surface of the protocerebrum, where
intrinsic MB axons are finally segregated into three distinct sets of lobes
(Fig. 2A). The
lobe
extends medially toward the midline and consists of the axons derived from the
MB neurons (
neurons) that are born before the mid-3rd instar stage
(Lee et al., 1999
). The
' and ß' lobes, which project perpendicularly from
each other, are derived from bifurcation of the axons of late larval-born MB
neurons (
'/ß' neurons)
(Lee et al., 1999
). The other
pair of perpendicular lobes, the
and ß lobes, are composed of
segregated axonal branches that are derived from pupal-born MB neurons
(
/ß neurons) (Lee et al.,
1999
). As implicated by the fact that newly deposited MB axons are
centrally localized, individual subtypes of axons are probably organized into
concentric longitudinal compartments based on orders of birth within the
peduncle and most lobe regions (Kurusu et
al., 2002
). In addition, four lineages simultaneously produce
identical MB axons that get fasciculated together way before reaching the
peduncle end, so distal parts of the peduncle as well as all the entire MB
lobes are fourfold structures (Ito et al.,
1997
; Yang et al.,
1995
). In contrast with what is known about MB axon fasciculation
patterns, very little has been learned regarding how MB dendrites are
spatially organized in the calyx, mainly because of a lack of recognizable
landmarks.
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To examine how numerous dendrites are organized in the MB calyx, we
selectively labeled various subsets of MB dendrites based on clonal origins
and/or cell types. We identified distinct clonal dendritic territories in the
adult calyx. Interestingly, various subtypes of MB dendrites contribute
differentially to different calycal regions. Single-neuron analysis further
revealed that pioneer /ß neurons acquire unique dendritic
elaboration patterns and consistently project their primary dendrites into a
small common space, the only fourfold adult calycal domain that receives
dendritic inputs from all four MB clonal units. In addition, analysis of
single neurons allowed us to identify distinct stage-specific dendritic
arborization patterns in the MB
neurons that regenerate dendrites
after pruning of larval processes during metamorphosis. Taken together, the
adult MB calyx is probably composed of multiple functionally distinct
compartments in which synaptic connections can be made between specific
subtypes of MB neurons and certain antennal lobe PNs.
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MATERIALS AND METHODS |
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Induction of flip-out clones
Induction of the Flipase activity was performed by heat shocking newly
hatched larvae, which carry Hs-FLP, UAS>rCD2,y+>mCD8-GFP, and one MB
GAL4, at 38°C for 30 minutes. Flip-out clones were examined with confocal
microscopy after immunostaining.
MARCM analysis of dendritic elaboration patterns of subtypes of MB
neurons
Single-cell/two-cell MARCM clones of ,
'/ß'
and
/ß neurons were generated by applying 10 minutes of heat shock
at various developmental stages as described previously
(Lee et al., 1999
). Around
pupal formation, organisms were re-synchronized using white pupae as the
reference point. A 10-minute heat shock was applied before or after formation
of white pupae at 2-hour intervals. MARCM clones were examined with confocal
microscopy after immunostaining.
Immunohistochemistry and confocal microscopy
Fly brains were fixed and subjected to immunostaining following the
procedures as described previously (Lee et
al., 1999). Primary antibodies used in this study include rat
anti-mouse CD8 mAb (1:100; Caltag), mouse anti-rat CD2 mAb (1:100; Caltag),
mouse anti-FAS II mAb 1D4 (1:50; a gift from C. Goodman), and mouse mAb nc82
that recognizes a synaptic antigen (1:20; a gift from E. Buchner). FITC- and
Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) were used at
1:100 and 1:500, respectively. Confocal images were collected using a Zeiss
LSM510 confocal microscope and processed with Adobe PhotoShop.
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RESULTS |
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Using UAS>rCD2,y+>mCD8-GFP as a reporter gene, one can label
GAL4-positive cells with either rCD2 or mCD8-GFP depending on whether the FRT
flip-out cassette is excised (Wong et al.,
2002). Therefore, in the presence of GAL4-OK107 that selectively
labels most MB neurons (Connolly et al.,
1996
), we could mark one clone of MB neurons differentially from
the other three clones by inducing excision of the FRT cassette in one of the
four MB neuroblasts at early developmental stages. Flip-out was induced
randomly in newly hatched larvae, and reporter gene expression patterns were
examined at the wandering larval stage or in adult flies. Our initial analysis
was focused on the brain lobes that display expression of mCD8-GFP in only one
clone of MB neurons. We found that one clone of MB dendrites can occupy the
entire calyx in wandering 3rd-instar larvae, as evidenced by complete
overlapping between the mCD8-GFP-positive dendrites and the rCD2-labeled
dendrites (Fig. 1A-C). This
observation indicates that the larval MB calyx is a perfect fourfold
structure. In contrast, after eclosion, the mCD8-GFP-positive dendrites that
are derived from a single clone fail to project throughout the entire calyx
and appear to occupy a smaller and distinct spatial domain, as compared with
the other three clones of rCD2-marked dendrites
(Fig. 1D-F). These results
suggest that the adult MB calyx, unlike its larval counterpart, exhibits
regional differences in the distribution of dendrites that are derived from
different clonal units.
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Identification of a small fourfold dendritic domain in the adult
calyx
Through analysis of series of coronal focal planes, we further identify a
small fourfold spatial domain that receives dendritic inputs from all of the
four MB clones at the adult stage (arrows in
Fig. 2C, parts g,h). This
unique fourfold dendritic domain corresponds to the most anterior tip of the
calyx and is probably equidistant from every clone. When one clone of MB
dendrites is specifically marked, regardless of its clonal identity, subsets
of the marked dendrites consistently reach and label a common dorsal-anterior
calycal region that is always shown on the most frontal coronal section of the
calyx (data not shown; similar to Fig.
3G). In addition, when two far-apart clonal units, for example the
AL and AM clones, are selectively labeled, we observe invading of dendritic
processes from distinct clones into each other's territories exclusively at
the dorsal-anterior spot (data not shown). It is apparent that only the
dorsal-anterior calycal subregion remains as a fourfold dendritic domain in
the adult MBs. In addition, we observe restriction of large degrees of mixing
of the MB dendrites among neighboring clones to the crescent area immediately
around the fourfold dendritic domain (Fig.
2C, part e; arrowhead in Fig.
3F), supporting possible presence of specific threefold dendritic
fields (see Discussion; Fig.
7).
Subsets of /ß dendrites, but not
dendrites, are
exuberantly mixed between neighboring clones
Each MB clone consists of a similar set of distinct types of neurons that
are sequentially generated from a neuroblast during development
(Lee et al., 1999). Given that
the adult calyx contains multiple clone-specific domains plus a fourfold
region shared by all the clones, we wondered whether distinct subtypes of MB
neurons elaborate their dendrites in different calycal subregions. With
GAL4-201Y (Yang et al., 1995
),
we selectively labeled all
neurons and a small number of late-born
/ß neurons in the mosaic MBs in which distinct clonal units are
differentially marked using the UAS>rCD2,y+>mCD8-GFP transgene. We found
that GAL4-201Y-positive dendrites extend throughout the clonal-dependent
regions (Fig. 3A-C), but fail
to reach the dorsal-anterior surface of the calyx such that the fourfold
dendritic domain is undetectable (data not shown). This result indicates that
neither
neurons nor late-born
/ß neurons normally project
their dendrites into the fourfold calycal subregion. In contrast, when
/ß neurons are selectively labeled using GAL4-c739
(Yang et al., 1995
), the
fourfold calycal region appears fully marked (arrowheads in
Fig. 3G-I), suggesting that
GAL4-201Y-negative
/ß neurons specifically mediate formation of
the fourfold dendritic domain in the adult calyx.
In addition to making no contribution to the fourfold dendritic domain, GAL4-201Y-positive dendrites seldom cross the clonal boundaries, as evidenced by presence of minimal mixing between differentially labeled clones of GAL4-201Y-positive dendrites (Fig. 3A-C). In contrast, significant overlap exists even outside the fourfold dendritic domain when two neighboring clones of GAL4-c739-positive dendrites are differentially labeled (arrowheads in Fig. 3D-F). Interestingly, GAL4-739-labeled dendrites, but not GAL4-201Y-positive dendrites, are selectively concentrated in five separate spacesfour clone-specific domains plus one shared domain in which dendrites of various clonal origins are significantly mixed (Fig.3D-F). These phenomena again suggest that distinct subtypes of MB dendrites are differentially distributed in the adult calyx.
/ß neurons acquire birth order-dependent dendritic
elaboration patterns and pioneer
/ß neurons contribute to the only
fourfold dendritic domain within the adult calyx
To identify which subtype of MB neurons elaborates their dendrites in the
fourfold calycal region, we re-examined individual MB neurons' dendritic
branching/projecting patterns. Using GAL4-OK107 in the MARCM system
(Lee and Luo, 1999), we had
systematically labeled single MB neurons based on their birth orders by
inducing mitotic recombination at different developmental stages
(Lee et al., 1999
). We reviewed
multiple isolated single-cell clones of MB neurons that were generated at
various larval and pupal stages. Although individual neurons acquire
morphologically variable dendritic branches, quantitative analysis reveals
several common features in their dendrite elaboration patterns
(Fig. 4). First, all the
neurons have three to six primary dendritic branches that often lack secondary
branches and directly end with `claw-like structures' (referring to clustered
fine dendritic arbors; e.g. arrows in Fig.
4). Second, primary branching points are spaced regularly with an
average interval of
7 µm along individual primary neurites. Third,
individual dendrites rarely cross each other even in the presence of multiple
single-cell clones (e.g. Fig.
6E,F). All the
dendrites appear to be distributed locally, making them unlikely to be
involved in formation of the fourfold dendritic domain.
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Pruning of larval dendrites allows neurons to alter dendritic
elaboration patterns during metamorphosis
Metamorphosis of MB neurons involves pruning of all larval
dendritic processes as well as larval-specific axonal branches
(Lee et al., 1999
). Although
the entire larval calyx, mainly consisting of
dendrites, is a fourfold
structure,
neurons are not involved in formation of the fourfold
dendritic domain in the adult calyx. To elucidate how pruning of larval
dendrites contributes to
neurons' final, mature dendritic arborization
patterns, we examined whether
neurons acquire different dendrite
branching/projecting patterns at the adult stage if their dendrites fail to be
pruned during metamorphosis. Single-cell clones of MB
neurons that are
homozygous for a loss-of-function mutation in ultraspiracle, baboon
or dSmad2, have been shown to be defective in pruning of both larval
dendrites and axonal branches at the pupal stage
(Lee et al., 2000
;
Zheng et al., 2003
). We
characterized dendritic elaboration patterns in isolated single-cell clones of
such mutant
neurons, and detected similar abnormal dendritic
arborization patterns in all remodeling-defective mutant
neurons
(Fig. 6A-C). First, all
wild-type
neurons have three or more primary dendritic branches
(average=4.9±1.1; n=12)
(Fig. 6D-F), whereas 50% of
mutant neurons have only one primary branch (average=1.4±0.5;
n=20) (Fig. 6A-C).
Second, primary branching points are distributed widely over the primary
neurites in wild-type
neurons (Fig.
6D-F), whereas most mutant dendrites appear to originate within a
narrow circumferential region above the equator of the adult calyx
(Fig. 6A-C). Third, wild-type
dendrites usually elaborate in a non-overlapping manner and end with distinct
claw-like structures; and individual terminal branches appear to occupy
discrete territories of their own (Fig.
6D-F). Therefore, when only one or two
neurons within each
of the four MB clonal units are simultaneously labeled, their dendrites are
widely distributed and roughly outline the entire calyx
(Fig. 6F). In contrast, mutant
dendrites often project toward a common area and tangle with one another
(arrow in Fig. 6B). As a
consequence, multiple mutant dendrites that are derived from different clonal
units become extensively intermingled and occupy a relatively restricted space
close to the central anterior surface of the calyx, forming an ectopic
fourfold dendritic domain (arrow in Fig.
6C).
Interestingly, primary branching patterns are basically preserved between
larval neurons and the adult
neurons that have retained their
larval processes into the adult stage. Single-cell analysis of larval
neurons reveals that individual
neurites generate one to two primary
dendritic branches over a narrow 7-µm range at the larval stage
(Fig. 6G-I). However, larval
dendrites undergo immediate arborization
(Fig. 6G-I); but retained
larval dendrites in the mutant
neurons remain relatively unelaborated
until reaching the ectopic fourfold dendritic domain at the adult stage
(Fig. 6A-C). These phenomena
underscore the physiological significance for pruning of larval dendrites in
establishing MB
neurons' mature dendritic arborization/distribution
patterns.
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DISCUSSION |
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Single-neuron analysis further reveals that pupal-born, adult-specific
/ß neurons acquire birth order-dependent dendritic
elaboration/distribution patterns. Pioneer
/ß neurons that are
born around pupal formation consistently send one long dendritic process along
the upper surface of the calyx into the fourfold anterior calycal tip.
Interestingly, the same neurons give rise to short branches that roughly cover
the calyx base. These pioneer
/ß dendrites, together with primary
neurites, outline the entire adult calyx. Given that larval dendrites are
largely pruned when pioneer
/ß neurons are born
(Lee et al., 1999
), these
first-derived adult MB dendrites might play important roles in guiding
elaboration of later-derived dendritic processes. Pioneer
/ß
neurons also acquire unique axonal branches that are probably involved in
initial formation of the MB
and ß lobes. In addition, as
implicated from analysis of subsequently born early
/ß neurons,
sequentially derived
/ß dendrites end shorter and shorter,
potentially forming concentric layers of dendritic termini outside the
fourfold domain. Partitioning of the calyces into several concentric
subdivisions has been documented in other insects with well-developed MBs
(Gronenberg, 2001
;
Strausfeld and Li, 1999
). In
contrast with orderly organization of the
/ß dendrites, no special
pattern has been observed for the distribution of
dendrites. In the
Drosophila brain,
/ß neurons are selectively involved in
olfactory associative long-term memory
(Pascual and Preat, 2001
). It
remains to be examined whether and how orderly organization of the
/ß dendrites contributes to processing and integration of distinct
olfactory information in the MBs.
Patterns of elaboration and distribution of adult MB dendrites are probably
established through sequential deposition of different subtypes of dendrites.
This scenario may explain why /ß neurons, but not
neurons,
acquire distinct dendrite elaboration/distribution patterns depending on when
they are born. Although
and
/ß neurons are both
sequentially generated during development, all
neurons simultaneously
remodel their dendritic processes during early metamorphosis whereas
/ß neurons sequentially deposit their dendrites through the pupal
stage (Lee et al., 1999
). Given
that all mature-looking larval dendrites are pruned around pupal formation
(Lee et al., 1999
), pioneer
adult MB dendrites are probably derived from the first-born
/ß
neurons whose dendritic processes, in fact, outline the adult calyx. In
addition, regeneration of
dendrites occurs around 24 hours after pupal
formation (Lee et al., 1999
),
which roughly coincides with the transition in
/ß neurons'
dendritic elaboration from birth order-dependent unique patterns to the
universal adult-specific pattern that is characterized by the presence of
multiple, regularly separated, simple primary branches. Interestingly,
late-born
/ß neurons appear to restrict their dendritic growth to
small individual clone-specific regions, which may simply be because of the
fact that limited space is left after full elaboration of adult
dendrites. All of these phenomena support that the adult MB calyx consists of
region-specific stereotyped dendrites, orderly organization of which possibly
involves sequential elaboration of distinct types of adult dendrites during
the pupal stage.
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ACKNOWLEDGMENTS |
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