A Complete Developmental Sequence of a Drosophila
Neuronal Lineage as Revealed by Twin-Spot MARCM
Hung-Hsiang Yu1., Chih-Fei Kao2., Yisheng He2, Peng Ding2, Jui-Chun Kao2, Tzumin Lee1,2*
1Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, United States of America, 2Department of Neurobiology, University of
Massachusetts, Worcester, Massachusetts, United States of America
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns
from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates
the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called
twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different
colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth.
Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common
neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the
sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During
embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL
are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast
resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order
strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic
progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the
same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated.
These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not
only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure
and function analysis of the brain.
Citation: Yu H-H, Kao C-F, He Y, Ding P, Kao J-C, et al. (2010) A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot
MARCM. PLoS Biol 8(8): e1000461. doi:10.1371/journal.pbio.1000461
Academic Editor: Hugo J. Bellen, Baylor College of Medicine, United States of America
Received April 7, 2010; Accepted July 13, 2010; Published August 24, 2010
Copyright: ? 2010 Yu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Howard Hughes Medical Institute and National Institutes of Health (NIH grant MH080739). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: adPN, anterodorsal projection neuron; AL, antennal lobe; CNS, central nervous system; FLP, flipase; GMC, ganglion mother cell; HS, heat shock;
iACT, inner antennocerebral tract; LH, lateral horn; LN, local interneuron; MARCM, mosaic analysis with a repressible cell marker; MB, mushroom body; NB,
neuroblast; ORN, olfactory receptor neuron; PN, projection neuron.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
The brain consists of a great diversity of neurons derived from
only a limited number of progenitors, called neuroblasts (NBs) [1–4].
Most NBs generate multiple neuron types [1,5–7]. Notably, specific
neurons are made by specific NBs at specific times of development,
suggesting stereotyped patterns of neurogenesis [6–9]. High-
resolution cell lineage analysis permits systematic identification of
neuron types by resolving every single neuron in a neuronal lineage.
Determination of neuron types based on their developmental origin
will not only reveal the circuitry of the brain but also illustrate how a
complex brain develops.
The clonal nature of brain development is particularly evident in
organisms where neurons of the same clonal origin remain clustered
in the mature brain [10–12]. The ability to recognize individual
clones and follow their development has shed much light on the
development and organization of the Drosophila central nervous
system (CNS) [6,7,11,12], in which NBs are individually identifiable
[3,4,13–15]. They acquire region-specific cell fates and generate
progeny whose projections are characteristic to each lineage
[6,7,12,16]. Neurons of a lineage derive sequentially: a given NB
repeatedly undergoes asymmetric cell division to renew itself and
produce a ganglion mother cell (GMC), which divides once to
produce two mature neurons . Such sister cells derived from a
GMC may acquire distinct fates due to differential Notch signaling
 andarefurther organizedaccordingto their hemilineage origin
[19,20]. Thus, most neuronal lineages consist of two hemilineages
with distinct trajectories, and many grossly homogeneous lineages
actually exist as lone hemilineages because their counterparts die
during development through apoptosis [19–21].
A mature brain, comprised of a huge repertoire of diverse
neurons, requires the production of multiple neuron types per
hemilineage [6,7,16,22]. The neurogenic diversity of holometabo-
lous insects arises in two waves : first, embryonically, most NBs
produce primary neurons for wiring of larval circuitry , which
may remodel during metamorphosis to contribute to the adult
circuitry [24–26]; second, NBs generate adult-specific secondary
neurons throughout larval development [6,7,12,16]. A complete
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neuronal lineage can thus be divided into two discrete blocks, with
multiple neuron types arising in a stereotyped pattern within each
developmental epoch [7,16,26]. Birthdating of identifiable primary
neurons in the embryonic ventral ganglion has revealed that unique
neurons, at least for the first-few-born neurons, within a clone
originate in an invariant sequence . Diverse secondary neurons
of the same hemilineage also derive sequentially in non-overlapping
windows [7,16]. Notably, the number of distinguishable cell types
that derive in a given window may vary drastically in different
lineages [6,7,16,28]. Thus, distinct NBs produce multiple neuron
types in different lineage-specific temporal patterns [6,7,16,28], and
sister hemilineages may even alter temporal identity at different
tempos . To identify all neuron types in such stereotyped
lineages, one should identify every single neuron of each
hemilineage based on the neuronal birth order.
An improved MARCM (Mosaic Analysis with a Repressible
Cell Marker) technique, called twin-spot MARCM, permits high-
resolution cell lineage analysis . Following mitotic recombi-
nation, twin-spot MARCM labels sister clones in distinct colors in
otherwise unstained tissue. In a typical neuronal lineage, a twin-
spot MARCM clone reveals two populations of cells: one or two
neurons derived from the GMC paired with all of the later-born
neurons in the lineage, which is labeled as the NB clone (e.g.,
Figure 1A). Counting the cell number of an NB clone reveals the
temporal position of its paired neuron(s) along the lineage (e.g.,
Figure 1A–C). Also, analysis of NB clones of all sizes in a
stereotyped lineage should reveal the order in which the post-
mitotic neurons of the lineage have been derived. Thus, a
complete description of neuron composition of the Drosophila brain
can be reached by identifying every single neuron in all lineages.
Such analysis will also uncover all neuronal trajectories and
elucidate the number of the same type of neurons that have
Stereotyped lineages underlie the development of the Drosophila
antennal lobe (AL), where a topographic map of olfaction is
and the AL projection neurons (PNs) (Figure 1D) [7,30–33]. There
are about 50 glomeruli in the adult AL (Figure 1E–H) .All ORNs
expressing the same odorant receptor project to the same glomerulus,
where they synapse with PNs; ORNs expressing different odorant
receptors project to distinct glomeruli [32,35–39]. Many PNs, like
ORNs, target only one AL glomerulus [22,33]. PNs send axons to
higher brain centers, including the mushroom body (MB) and the
lateral horn (LH) (Figure 1D) [22,33]. Distinct PNs further acquire
different characteristic patterns of axon projections [22,33]. Following
the trajectories of PNs that connect with distinct ORNs has started to
unravel how different olfactory inputs might be processed differen-
tially to govern diverse organismal behaviors . However, in
contrast with a near-complete description of ORNs [38,39], the
uniglomerular PNs of several AL glomeruli, if they exist, remain to be
identified [22,26,33]. In addition, there possibly exist diverse types of
to higher brain centers [22,29,40]. Therefore, the olfactory
topographic map of the adult AL will not be complete until all PN
types have been identified and counted.
Here we determined every single neuron in an AL PN lineage
through analysis of numerous twin-spot MARCM clones. We
uncovered 15 additional PN types, including five polyglomerular
types of PNs in the otherwise pure uniglomerular lineage; these
distinct PNs are born in an invariant sequence. Notably, the NB
alters temporal identity following each embryonic division and
yields 18 types of PNs during its brief production of primary
neurons. In contrast, only 22 morphologically distinguishable
types of PNs derive from the many more secondary neurons
generated by the same NB. Furthermore, these larval-born multi-
neuronal cell types show specific cell counts, suggesting the tightly
regulated fate of individual neurons chronologically as well as
spatially and supporting the functional significance of these
neurons. This is the first study, to our knowledge, to completely
describe the neuron composition of a neural lineage, and this also
underscores the importance of deciphering individual neurons in
all lineages to elucidate the brain development and function.
Strategies for Sequencing the AL PN Lineage That Can Be
Selectively Targeted by GAL4-GH146 and acj6-GAL4
Three AL PN lineages have been partially characterized
[7,22,26,29,33,41]. Among them, the anterodorsal PN (adPN)
lineage is best studied [7,19,22,26,33]. It exists as a lone
hemilineage and can be fully covered with acj6-GAL4 [19,29,42].
In addition, many adPNs are positive for GAL4-GH146. Twenty-
five types of uniglomerular PNs have been identified through
single-cell analysis of GH146-positive adPNs [7,22,33,36]. Distinct
adPNs derive in an invariant sequence [7,26]. However, their
birth order has not been completely resolved. In addition, the
number of neurons comprising the lineage is unknown. Finally,
GH146-negative adPNs have only been treated as a population
, and while some glomeruli innervated by these PNs have been
identified, the projection patterns of individual GH146-negative
adPNs remain undetermined.
To resolve the entire lineage based on neuronal birth order, we
first determined when most of the elusive GH146-negative adPNs
were born through analysis of adPN NB clones induced at different
times of development. Dual-expression-control MARCM allowed
us to label GH146-positive adPNs of the clones with LexA::GAD-
obtained similar numbers of GH146-negative cells among the dual-
expression-control MARCM clones, even when clones were
induced at the mid-3rd instar larval stage, labeling only the last
VA1lm-targeting GH146-positive adPN and subsequently born
neurons in the adPN NB clone (around 32 cells; n=5; Figure S1). In
A brain consists of numerous, potentially individually
unique neurons that derive from a limited number of
progenitors. It has been shown in various model organ-
isms that specific neurons arise in a lineage made by a
repeatedly renewing progenitor at specific times of
development. However, except in the worm C. elegans,
the stereotype of neural development has never been
examined in sufficient detail to account for every single
neuron derived from a common progenitor. Here we
applied a sophisticated genetic mosaic system to mark
single neurons in the adult Drosophila brain and simulta-
neously reveal in which lineage a targeted neuron had
arisen and when along the lineage it was made. We have
identified each neuron in a lineage of olfactory projection
neurons. There are a remarkable 40 types of neurons
within this lineage born over two epochs. Strikingly, the
birth order strictly dictates the fate of each post-mitotic
neuron, including the fate of programmed cell death, such
that every neuron type has a unique and invariant cell
count. Sequencing an entire neuronal lineage provides
definitive evidence for origin-dependent neuron type
specification. It further permits a systematic characteriza-
tion of neuron types for comprehensive circuitry mapping.
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these clones, six glomeruli were prominently marked by the
GH146-negative neurons (Figure S1B). These observations suggest
that most, if not all, GH146-negative adPNs derive after birth of
their GH146-positive siblings and that they innervate a distinct set
of glomeruli from the GH146-positive adPNs.
We supposed that use of GAL4-GH146 alone should allow us to
identify the majority of adPNs born prior to the mid-larval stage.
We therefore reserved acj6-GAL4, which has much more non-PN
expression, for the elucidation of the late larval development of the
adPN lineage and filling any gap present in the sequence of
GH146-positive adPNs. As described below, we first resolved the
GH146 part of the larval adPN lineage (Figure 2). We then
determined the later-derived GH146-negative adPNs using acj6-
GAL4 (Figure 3). Finally, we identified the embryonic-born adPNs
with GAL4-GH146, followed by acj6-GAL4 (Figure 4).
The Early Larval adPN NB Serially Generates 12
Uniglomerular PN Types and, in a Specific Interval, Some
To identify the larval-derived GH146-positive adPNs as well as
determine their birth order, we obtained numerous adPN NB
clones paired with distinct PNs following induction of mitotic
recombination at different times of larval development. The NB
clones may pair with one of the following 12 types of
uniglomerular PNs: DL1, DA3, DC2, D, VA3, DC3, VA1d, 1,
VM7, VM2, DM6, and VA1lm (in the order of disappearance
from the NB clones of decreasing sizes; see below; Figure 2A–M).
Four previously identified GH146-positive adPN types, including
DC1, DM4, VL2a, and VM4, were never detected among the
paired single-cell clones . These indicate production of specific
adPN types during early larval development.
We then determined if specific adPNs are derived from GMCs
born at specific windows of the lineage. Because distinct PNs target
different AL glomeruli, the offspring composition of a multi-
cellular adPN NB clone can be inferred based on the labeled AL
glomeruli. Thus, we determined which neurons remained to be
derived after birth of a particular PN by analyzing the offspring
composition of its paired NB clone. Using this method, we found
in every DL1-paired NB clone the presence of all the remaining 11
types of uniglomerular PNs, arguing that the birth of DL1 adPNs
precedes the others’ (the top panel of Figure 2A; n.50). The DL1
glomerulus was often targeted by the DL1 single neuron as well as
Figure 1. Examples of paired sister clones, the AL glomerular architecture, and a summary of adPNs. (A–C) GMC clones (magenta) pairs
with NB clones (green) of different sizes depending on when mitotic recombination occurred in a protracted lineage. Judging from the size of the
accompanying NB clone, one can determine when the GMC of a particular neuron was born in the lineage. One can therefore deduce in the adPN
lineage: the VM3-targeting neuron (magenta in [A]) born around the beginning, the 1-targeting neuron (magenta in [B]) derived in the middle, and
the DL2v-targeting neuron (magenta in [C]) made near the end. (D) A schematic illustration of an uniglomerular adPN (black) that connects one of the
50 or so AL glomeruli with the calyx (CA) of the mushroom body (MB) and the lateral horn (LH). The relative position of three populations of PNs, the
adPNs (blue), lPNs (red), and vPNs (orange), is also shown. (E–H) All identifiable glomeruli in the AL are shown in four anterior-to-posterior focal
sections. The glomerular targets of previously identified uniglomerular adPNs, lPNs, and vPNs are labeled in blue, red, and orange, respectively. The
glomerular targets of the uniglomerular adPNs identified in this study are shown in cyan. And the glomeruli with yellow labels have not yet found
their corresponding uniglomerular PNs. (I) adPNs identified previously and in this study are summarized. The adPNs with known birth order are
further arranged with respect to the lineage development. Fly brains were counterstained with nc82 mAb (blue) in this and all other figures, which
permits determination of glomerular identity in the AL. The scale bar in this and all other figures equals 10 mm.
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its paired NB clone, suggesting the presence of multiple DL1
adPNs (the middle panel of Figure 2A). Moreover, all the DL1 PNs
are born before the NB transits to make other PN types, since we
rarely detected the DL1 glomerulus in the NB clones paired with
other larval-derived adPN types (,1%, n.100; unpublished data;
such rare events possibly resulted from contamination with GMC-
derived single-cell clones). Annotation of the AL glomerular
targets of the adPN NB clones paired with each of the remaining
11 types of uniglomerular PNs subsequently revealed the order in
which the 12 types of GH146-positive adPNs derive during early
larval development (unpublished data; the analysis was done
similarly as shown in Table S1). Briefly, the DL1, DA3, DC2, D,
VA3, DC3, VA1d, 1, VM7, VM2, and DM6 glomerulus
correspondingly disappeared from the adPN NB clones paired
with the DA3, DC2, D, VA3, DC3, VA1d, 1, VM7, VM2, DM6,
and VA1lm PN (Figure 2B–G and 2I–M; n.4 for each). This
indicates an orderly derivation of 12 types of adPNs from a
Figure 2. Twelve types of early-larval-derived GH146-positive uniglomerular adPNs. Twin-spot MARCM clones of adPNs labeled with
GAL4-GH146 (A-M) or acj6-GAL4 (N). Top panels: composite confocal images of sister clones in the AL; middle panels: single focal sections of the AL
covering the glomerular targets of GMC progeny (magenta); bottom panels: axon projections of GMC progeny (magenta); islets in bottom panels:
axon projections of both GMC progeny (magenta) and its paired NB clone (green). Note each adPN type (magenta) consistently pairs with adPN NB
clones (green) of specific compositions. Analysis of NB clones revealed the 12 types of GH146-positive adPNs are made in an invariant sequence from
(A) to (M). And all the lone, unpaired NB clones (H), whose preceding GMC progeny probably die prematurely, were induced in the interval between
VA1d and 1 adPNs. The sequence of early-larval adPN neurogenesis is summarized in the bottom. In addition, there are multiple neurons per type, as
evidenced in middle panels that the glomerular target of GMC progeny can be co-labeled by its accompanying NB clone. For the lineage after VA1lm
PNs, one can visualize GH146-negative adPNs with acj6-GAL4 as revealed in (N) where the last VA1lm adPN pairs with a 32-neuron-containing NB
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In addition, we noticed the presence of unpaired NB clones that
consistently carry the last five types of GH146-positive adPNs
(Figure 2H; n=5). This indicates the absence of GMC progeny in
the interval between the generation of adPNs innervating VA1d
and 1. The same gap was also evident when we marked a similar
pool of adPN twin-spot clones using the pan-adPN driver, acj6-
GAL4 (unpublished data). Since no acj6-positive, GH146-negative
adPN was born in the same window, the post-mitotic neurons
made between the VA1d and 1 adPNs are probably programmed
to die. Taken together, the early larval adPN NB serially makes 12
uniglomerular PN types and, in a specific interval, some
prematurely lost cells.
Six Types of Uniglomerular PNs Plus Four Types of
Polyglomerular PNs Subsequently Derive After the
We next determined the subsequently derived GH146-negative
adPNs and their birth order through analysis of twin-spot clones
labeled with acj6-GAL4. The acj6-GAL4-labeled NB clones that
Figure 3. Ten types of late-larval-derived Acj6-positive adPNs. Late-larval-derived twin-spot clones labeled with acj6-GAL4. Top and middle
panels: composite confocal images of the AL showing single adPNs only or both single adPNs (magenta) and their paired NB clones (green); bottom
panels: the axon projections of single adPNs and in islets the projections of single adPNs (magenta) and their accompanying NB clones (green).
Analysis of NB clones paired with distinct adPNs revealed 10 additional adPN types are made in an invariant sequence as summarized in the bottom.
Note presence of four types of polyglomerular PNs that exhibit different patterns of AL elaboration while acquiring analogous axon projections in the
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paired with the last sibling of VA1lm-targeting adPNs, the end of
GH146-positive series, contain about 32 post-mitotic neurons
(Figure 2N; n=3). Similar cell numbers were obtained from
counting the GH146-negative cells among the dual-expression-
control MARCM clones (Figure S1; n=5). A distinct set of six AL
glomeruli, including VC4, VM5d, VM5v, VC3, DL2v, and DL2d,
were prominently labeled in the largest NB clones exclusively
negative for GAL4-GH146 (Figure 2N and Figure S1B). This
suggests probably six novel types of uniglomerular PNs are made
in the remaining adPN lineage.
Analysis of single-cell clones of acj6-positive, GH146-negative
adPNs generated in late larvae revealed the six types of
uniglomerular PNs (Figure 3A–D and 3I–J; n.4 for each).
Interestingly, we also identified PNs that spread neurites across
multiple glomeruli (Figure 3E–H; n.4 for each). Four distinct
polyglomerular patterns, tentatively referred to as poly[L1],
poly[L2], poly[L3], and poly[L4], could be detected (Figure 3E–
H). They innervate partially overlapping subsets of eight specific
AL glomeruli. The four patterns from poly[L1] to poly[L4],
respectively, cover: (1) parts of DC4, VC3, and VM4 (Figure 3E);
(2) VM4, VL2p, and regions posterior to medial ventral AL
(Figure 3F); (3) VL2p, VL2a, and DL1l (Figure 3G); and (4) parts
of VL2a, DL1l, DL2v, and DL2d (Figure 3H). Notably, such
polyglomerular adPNs exhibit similar trajectories in the LH
despite differences in the AL elaboration (bottom panels of
Figure 3E–H). This is in sharp contrast with their uniglomerular
siblings whose LH trajectories differ among PNs targeting distinct
glomeruli (e.g., Figures S4–S5) [7,22,33]. This might lead one to
wonder if different polyglomerular patterns may result from
developmental plasticity rather than being precisely prespecified.
Nonetheless, closer inspection permitted identification of these
four types of polyglomerular PNs even in NB clones (the middle
panels in Figure 3E–H). They disappeared again in an invariant
sequence from the adPN NB clones of decreasing sizes (see below),
demonstrating the presence of four polyglomerular PN types in the
otherwise uniglomerular PN lineage.
Analysis of adPN NB clones paired with distinct PNs, including
the six additional types of uniglomerular PNs and the four types of
polyglomerular PNs, further revealed that these 10 types of
GH146-negative PNs are consistently derived in the following
Figure 4. Eighteen types of embryonic-born adPNs. Embryonic-derived twin-spot clones labeled with GAL4-GH146 (A–V) or acj6-GAL4 (X–Z).
Top two panels: composite confocal images of the AL showing single adPNs (magenta) and the paired NB clones (green); middle panels: single focal
sections of the AL covering the glomerular targets of single adPNs; bottom two panels: axon projections of single adPNs (magenta) and the
accompanying NB clones (green). 22 types of GH146-labeled NB clones can be distinguished and are shown in the order of decreasing complexity
from (A) to (V). 15 of them pair with distinct adPNs while seven of them exist alone (no magenta labeling). The unpaired NB clones in (H), (J), and (M),
when labeled with acj6-GAL4, were paired with novel GH146-negative adPNs, including one additional type of polyglomerular PN (X–Z). The
sequence of embryonic-derived adPNs is shown in (W). Note presence of one neuron per type (except VM3-targeting PNs in [R] and [T]) in the lineage
of primary neurons, as evidenced by the glomerular targets of single adPNs (magenta) not innervated by their accompanying NB clones (green).
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specific order: VC4, VM5d, VM5v, VC3, poly[L1], poly[L2],
poly[L3], poly[L4], DL2v, and DL2d. Notably, the four types of
polyglomerular PNs are generated contiguously in the interval
between the production of VC3 and DL2v uniglomerular PNs
(Figure 3D–I). In other words, no gap exists in the GH146-
negative sublineage; unpaired adPN NB clones are observed only
in the earlier window when the adPN NB transits from making the
VA1d PNs to yielding the 1 PN (Figure 2G–I). Taken together,
through larval development, the adPN NB makes 22 types of AL
PNs, including 18 uniglomerular PN types and four polyglomer-
ular PN types.
18 Uniglomerular PNs, Targeting 17 Additional AL
Glomeruli, Plus One Polyglomerular PN Constitute the
Embryonic adPN Lineage
Following the determination of secondary neurons, we exam-
ined the primary neurons generated by the adPN NB. We first
determined the GH146-positive adPNs born in embryos. Clones
were induced during embryogenesis and labeled by twin-spot
MARCM with GAL4-GH146. We uncovered 14 additional types
of uniglomerular PNs through analysis of embryonic-derived,
single-cell clones of adPNs. They innervate one of 14 adult AL
glomeruli, including DP1m, VL2p, DA4l, VM4, VA6, VL2a,
DC1, VA7l, VA2, DM4, DL5, DM3, VM3, and DL4 (in the order
of disappearance from the NB clones of decreasing sizes; see
below; Figure 4A–V and Figure S2; Table S1). We further
obtained the adPN NB clones that pair with each of the 14 types of
primary neurons (Figure 4B, 4D, 4F–G, 4I, 4K–L, and 4N–U).
Intriguingly, the embryonic-derived adPN NB clones, except those
paired with VM3-targeting adPNs (Figure 4R and 4T), never
innervated the same glomerulus labeled by the GMC side of twin
spots. This indicates presence of only one adPN for the majority of
primary-neuron-targeted glomeruli. This stands in great contrast
to the larval-derived adPN types that consistently exist in multiple-
cell groups, as evidenced by their glomerular targets often being
co-labeled by both NB and GMC sides of twin spots (Figures 2–3).
We then analyzed the detailed glomerular innervation patterns
of the NB clones. Notably, the 14 glomeruli targeted by the
primary neurons disappeared in a stereotyped order as the clone
size decreased. This reveals derivation of distinct primary neurons
in an invariant sequence as well. It further shows production of
VM3 adPNs in two windows separated by the birth of the DM3
adPN (Figure 4R and 4T). There should be only one VM3 adPN
born after the DM3 adPN, since the VM3 glomerulus was never
co-labeled by both sides of twin spots after birth of the DM3 adPN
(Figure 4S–T). Indeed, a lone VM3 adPN consistently innervates
the VM3 glomerulus in coarse patches (the middle panel of
Figure 4T). Notably, the VM3 glomerulus was fully tiled upon co-
labeling by an earlier-derived VM3 adPN within the accompany-
ing NB clone (the middle panel of Figure 4R). This suggests
presence of only one VM3 adPN born before the DM3 adPN as
well. Besides innervating the same glomerulus in complementary
patches, they exhibit similar patterns of axon arborization in the
LH (Figure 4R and 4T). This argues for the presence of two
indistinguishable VM3 adPNs, despite their derivation in distinct
windows. In sum, the embryonic adPN NB makes 15 GH146-
positive uniglomerular PNs in the following order: DP1m, VL2p,
DA4l, VM4, VA6, VL2a, DC1, VA7l, VA2, DM4, DL5, VM3(a),
DM3, VM3(b), and DL4.
In addition, we obtained seven classes of unpaired NB clones
that show specific patterns of glomerular innervation, suggesting
the presence of some GH146-negative primary neurons and/or
premature loss of certain primary neurons (Figure 4A, 4C, 4E, 4H,
4J, 4M, and 4V). Analysis of twin-spot adPN clones marked with
acj6-GAL4 subsequently allowed us to identify two additional
uniglomerular PNs and one polyglomerular PN that fill three of
the seven gaps in the GH146-positive primary neuron sequence
(Figure 4X–Z; Table S2). The VM6+VP1 PN lies in the gap
between the VM4 adPN and the VA6 adPN (Figure 4X); the DL6
PN resides between VA6 and VL2a (Figure 4Y); and the
polyglomerular PN derives in the interval between DC1 and
VA7l (Figure 4Z).
Despite use of the pan-adPN driver acj6-GAL4, we obtained two
classes of unpaired NB clones (Figure 4A, 4V and unpublished
data; Table S2). These occurred following mitotic recombination
at the beginning of the lineage and at the end of primary neuron
production, respectively. The lone largest clones were probably hit
during the birth of the adPN NB, and their sister clones could
reside outside the CNS (Figure 4A). In contrast, the lone NB
clones that exclusively consist of the entire secondary lineage
should pair with the last-born primary neuron, absence of which
indicates another neuronal loss in the protracted adPN hemi-
lineage (Figure 4V).
The remaining two gaps in the sequence of GH146-positive
adPNs were unresolved following analysis of all the acj6-labeled
NB clones induced during embryogenesis (Table S2). These should
be occupied by the 2nd and 4th sibling, respectively, but we did
not obtain acj6-labeled NB clones hit during the birth of their
precursors. Therefore, the fate of these post-mitotic neurons
remains to be determined. Nonetheless, detailed analysis of several
acj6-labeled, full-sized NB clones (Figure S3A–S3D) allowed us to
detect VP3 as another glomerular target of adPNs (Figure S3D).
We could also obtain single-cell clones of VP3-targeting PNs when
clones were labeled with acj6-GAL4 (Figure S3E). Further, the VP3
adPN was absent from the NB clones paired with the fifth or any
later-derived adPN (unpublished data). Since no additional
uniglomerular adPN could be found, the VP3 adPN and possibly
another premature-lost neuron might account for the final two
gaps in the otherwise completely resolved adPN sequence.
Taken together, the embryonic adPN NB invariantly makes 19
viable PNs, including 18 uniglomerular PNs of 17 types and one
polyglomerular PN, from a possible set of 21 asymmetric cell
divisions. There is minimal cellular redundancy with one neuron
per glomerular target except the VM3 glomerulus. This is in great
contrast with the secondary neurons of the same lineage, which
outnumber the primary neurons by 3- to 4-fold but only add a
comparable number of PN types (22 versus 18) to the mature
Multi-Cellular PN Types Show Stereotyped Cell Counts
Because the AL glomeruli innervated by primary versus
secondary neurons operate through solo or multiple PNs, we
wondered if fixed cell counts exist for the multi-cellular adPN types
composed of secondary neurons. To answer this question, one
needs to count the cells of NB clones. We first analyzed GH146-
labeled NB clones and started with the ones homogeneously
consisting of VA1lm PNs (the last-derived GH146-positive adPN
type; Figure 5A; Table S3). All the NB clones that paired with the
last DM6 sibling (the preceding adPN type) and thus carrying an
entire set of VA1lm adPNs had five cell bodies. This indicates that
there are always five VA1lm PNs made by the adPN NB. And the
NB clones paired with the last VM2 sibling (the adPN type
preceding DM6) possessed eight neurons that include five VA1lm
and three DM6 uniglomerular PNs (Figure 5B; Table S3). In this
way, we worked backward to determine the cell numbers for late-
to early-derived adPN types. Invariant cell counts were obtained
for the majority of NB clones paired with the last sibling of six
contiguous PN types, which allowed us to deduce that the adPN
Comprehensive Lineage Analysis by Twin-Spot MARCM
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NB consistently makes 5 VA1lm, 3 DM6, 2 VM2, 3 VM7, 2 1,
and 4 VA1d PNs (Figure 5A–G; Table S3).
Analysis of acj6-labeled NB clones using the same strategy
revealed invariant cell fates for the last-born 21 adPNs as well.
After making the last VC3 uniglomerular PN, the NB serially
yielded 10 polyglomerular, 4 DL2v, and 6 DL2d PNs as the
lineage ends by pupation (Figure 5K–M; Table S4). The
polyglomerular patterns of innervation could only be unambigu-
ously discerned in single-cell clones, preventing further cell counts
for specific polyglomerular types. In conclusion, the adPN NB not
only makes specific types of AL PNs but also generates a fixed
number of neurons for each PN type.
The AL receives odorant inputs from ORNs that reside in two
peripheral appendixes, the antennae and maxillary pulps.
Identification and characterization of various types of PNs and
local interneurons (LNs) in the AL has enhanced our understand-
ing of how the olfactory information is relayed and integrated in
the Drosophila brain [7,22,26,29,33,41,44]. Given the stereotypy of
neural development, comprehensive cell lineage analysis, which is
made possible by twin-spot MARCM , should allow one to
identify all AL PNs and LNs systematically. Here we have
determined every single neuron within one of five AL lineages (see
below for the description of these five AL lineages). Forty types of
AL PNs derive in an invariant sequence in the adPN lineage
(Figures 2–4) and diverse PN types further show distinct, specific
cell counts (Figure 5). These argue that the fate of individual
neurons in the adPN lineage is tightly regulated during
development and possibly hint at the functional significance of
these neurons in the olfactory circuit.
Clonal analysis using ubiquitous drivers has allowed us to
uncover five neuronal lineages that generate neurons predomi-
nantly innervating the AL (Yu and Lee, unpublished observation).
These include the previously reported adPN, lALN, and vPN
lineages [7,41], in addition to the vLN and lvPN lineages (Yu and
Lee, unpublished observation). The vLN lineage generates LNs
with cell bodies located ventral to the AL, while the adPN, lvPN,
and vPN lineages produce PNs with cell bodies residing
Figure 5. Distinct cell counts in different adPN types. Twin-spot clones labeled with GAL4-GH146 (A–G) or acj6-GAL4 (K–M). Upper panels:
composite confocal images of sister clones in the AL; lower panels: single focal sections showing no innervation of the magenta glomeruli by the
green NB clones, indicating clones derived during birth of the last sibling of the preceding adPN type. The clones shown in (A) and illustrated in (H)
reveal the adPN NB makes five VA1lm-targeting PNs following derivation of the last DM6-targeting PN. Illustrations of lineage development for
additional twin-spot clones are shown in (I), (J), and (N) to (P). Invariant cell counts were obtained for the majority of NB clones paired with the last
sibling of the preceding adPN type (see Tables S3 and S4). These support production of a fixed number of neurons for each multi-cellular adPN type,
as summarized in the bottom.
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anterodorsal, lateroventral, and ventral to the AL, respectively. By
contrast, the lALN lineage makes both LNs and PNs with cell
bodies clustered lateral to the AL. Single-cell analysis has so far
revealed that, excepting the lALN lineage, the remaining four
lineages are homogeneously composed of AL neurons. By contrast,
characterization of about 85 additional central brain lineages
revealed only one lineage that shows sparse AL innervation (Yu
and Lee, unpublished observation). These observations suggest
that a specific set of neuronal lineages are selectively devoted to the
AL development. Comprehensive lineage analysis to identify every
single neuron within the five AL lineages should facilitate the
reconstruction of neural circuitry in the primary olfactory center.
The AL relays olfactory information from ORNs to higher
brain centers primarily through PNs that connect 52 AL glomeruli
to the MB calyx and the LH. Assuming the presence of
uniglomerular PNs for all AL glomeruli, 52 types of uniglomerular
PNs are anticipated. Complete sequencing of the adPN lineage
reveals 35 of them, and analysis of GH146-positive PNs in the
lALN lineage has shown 12 additional types of uniglomerular PNs
. Notably, we identified a GH146-negative adPN innervating
a previously unreported glomerulus (DL6; Figure 1Y) while finding
another GH146-negative adPN occupying two vaguely separate
glomeruli (VM6+VP1; Figure 1X) whose input(s) remains
unknown . We have thus modified the list of 52 AL glomeruli
by adding DL6 and combining VM6 with VP1 (Figure 4X–Y),
leaving the total number of AL glomeruli unchanged. Note that
the ‘‘DL6’’ uniglomerular PN first mentioned in an earlier
MARCM study of GH146-positive adPNs innervates DL4 rather
than a previously unreported glomerulus . Our study leaves
five AL glomeruli, DA4m, V, DC4, VP2, and VL1, whose
corresponding MB/LH-targeting uniglomerular PNs remain to be
uncovered. The missing types of uniglomerular PNs should exist:
with the exception of VP2, the olfactory inputs for these glomeruli
are known and comparable to those innervating other AL
glomeruli [38,39]. Identifying the entire set of uniglomerular
PNs and further determining their connectivity in the LH will help
elucidate how odors govern organismal behavior.
Besides uncovering 10 previously unreported uniglomerular PN
types, sequencing the entire adPN lineage has unexpectedly led to
the discovery of a new class of polyglomerular PNs. Only a few
polyglomerular PN types had been identified previously [22,29].
They differ from uniglomerular PNs not only in the pattern of
glomerular innervation but also in the trajectory of their axons. In
contrast with the above 47 types of uniglomerular PNs that extend
axons through the iACT and consistently target both the MB and
LH (Figures 2–4) [22,33], the axons of earlier identified poly-
glomerular PNs take distinct paths and innervate diverse targets,
of polyglomerular adPNs that we identify here navigate through the
the MB and LH. However, they rarely made branches or synaptic
boutons within the MB calyx (Table S5). Their axons appear to
Figures 3E–H and 4Z). Given the distinction, polyglomerular PNs
probably serve different functions from the much better known
uniglomerular PNs. Notably, the four contiguously born polyglo-
merular adPN types exhibit dendritic tiling within the AL and
further acquire an indistinguishable axon trajectory in the LH
(Figure 3E–H). Interestingly, their glomerular targets selectively
receive inputs from the coeloconic type of sensilla . Although the
that these polyglomerular PNs jointly modulate some innate
behavior(s) in response to specific olfactory inputs.
Sequencing an entire lineage further revealed that the cell
numbers of specific neuron types are tightly regulated (Figures 4–
5). Embryonic-born adPN types have only a single neuron, with
the exception of two neurons innervating VM3 (Figure 4). In
contrast, all the adPN types derived after larval hatching are made
up of multiple neurons (Figures 2–3). No difference in the size of
glomeruli or quality of the olfactory input could be discerned
between the single- and multi-PN glomeruli, and glomeruli instead
show considerable variety: a lone uniglomerular PN can densely
fill a large glomerulus (e.g., DP1m and VA2 in Figure 4B and 4O),
while some small glomeruli are tiled by multiple PNs (e.g., DA3,
VM2, and 1 in Figure 2B, 2I, and 2K). However, among the
multi-PN glomeruli, the glomerular size does roughly correlate
with its number of uniglomerular PNs. For example, the VA1lm
and VA1d glomeruli are respectively innervated by five and four
adPNs and are much more prominent than the VM2 and 1
glomeruli, both tiled by only two adPNs (Figure 5). Thus, the role
of added PNs innervating a given glomerulus is not simply to allow
for dendritic tiling and may play a role in mechanisms of olfactory
We have further demonstrated the longstanding observation
that adPNs possess distinct stereotyped axonal projection patterns
within the LH (Figures S4–S5) [22,26,33]. However, we have also
shown that PNs with the same glomerular target exhibit nearly
indistinguishable patterns of axon arborization. When two adPNs
were differentially marked as single-cell clones by twin-spot
MARCM, we frequently observe among neurons targeting the
same glomerulus co-migration of neurites and co-localization of
bouton-like structures in the LH (Figure S4A–S4F compared with
S4G–S4H). The same phenomena apply to the two separately
derived VM3-targeting adPNs, arguing that contiguous birth is
not a prerequisite for such coordinated projections (Figure 4R,
4T; Figure S5G and S5I). These data support the presence of
multiple anatomically equivalent neurons per larval-derived
Finally, neuronal birth order strictly dictates the fate of each
post-mitotic neuron. This mechanism of predetermined cell
fates nicely explains the observed stereotyped development of
the adPN lineage, including uniglomerular PNs targeting one of
the 52 AL glomeruli (Figures 2–4), premature cell loss
(Figure 2H), and polyglomerular PNs that tile specific glomeruli
in an invariant pattern while sending axons to the same
probable target(s) (Figure 3E–H). Development clearly guides
how neural circuitry is built, which, in turn, shapes development
through evolution. Like other primary neurons, most adPNs
born during embryogenesis are individually unique and have
elaborated exuberantly enough to serve the functions demand-
ing multiple secondary neurons
secondary neurons are made in blocks during larval develop-
ment with multiple cells acquiring the same fate within each
block (Figures 2–3). Notably, post-embryonic NBs alter their
temporal identity in lineage-specific patterns. Such develop-
mental programs not only control neuron types but also confer
cell counts to each neuron type and constrain how the circuitry
is built. To elucidate why AL PN numbers, including those of
determination of the physiological consequences of perturbing
cell numbers. To comprehensively determine neuron types
based on developmental origin should reveal the organization of
the entire brain, and to investigate how stereotyped lineages are
made and further gain the ability to engineer their development
will advance our understanding of the ultimate mechanism of
function of the brain.
(Figure 4). In contrast,
Comprehensive Lineage Analysis by Twin-Spot MARCM
PLoS Biology | www.plosbiology.org9 August 2010 | Volume 8 | Issue 8 | e1000461
Materials and Methods
The fly strains used in this study were: (1) Acj6-GAL4 ; (2)
FRT40A,UAS-Cd2::rfp,UAS-gfp-Mir/CyO,Y ; (3) FRT40A, UAS-
Cd8::gfp,UAS-Cd2-Mir/CyO,Y ; (4) FRT40A,UAS-Cd8::gfp,UAS-
Cd2-Mir,GAL4-GH146/CyO,Y; (5) FRT40A,UAS-Cd8::gfp,lexAop-Cd2-
gfp,LexA::GAD-GH146/CyO,Y; (6) hs-FLP ; (7) hs-FLP
; and (8) FRT40A,tubP-GAL80/CyO,Y .
Clonal Analysis with Dual-Expression-Control MARCM
and Twin-Spot MARCM
The generation, dissection, immunostaining, and mounting of
mosaic clones of adult brains have been described . For dual-
expression-control MARCM experiments, mosaic clones were
induced using hs-FLP  at early larval and mid-3rd instar larval
stages by heat-shock for 20 and 35 min, respectively. Determina-
tion of the birth order of PNs in Figures 2–4 is based on the cell
number and neuronal composition of their paired NB clones, so
samples were only roughly synchronized for heat shock at various
later time points in twin-spot MARCM experiments. In short,
mosaic clones of embryonic-born PNs were generated with hs-
FLP by collecting embryos for 18 h in vials and following
with heat-shock for 10 min. Mosaic clones of larval-derived PNs
were induced with either hs-FLP or hs-FLP by collecting
embryos for 12 h in vials and heat-shock for 20–40 min at
different developmental stages (every half day from 0.5 d after
embryo collection to puparium formation). For example, DL1
paired with its NB clone in Figure 2A can be generated at 0.5–2 d
after embryo collection. DA3 paired with its NB clone in Figure 2B
can be generated at 2 d after embryo collection. VA1lm paired
with its NB clone in Figure 2M can be generated at 4 d after
embryo collection. DC2, D, VA3, DC3, VA1d, 1, VM7, VM2,
and DM6 paired with their NB clones in Figure 2C–O can be
generated between 2.5 d to 4 d after embryo collection. VC4,
VM5d, VM5v, VC3, poly[L1], poly[L2], poly[L3], poly[L4],
DL2v, and DL2d paired with their NB clones in Figure 3 can be
generated between 4 d to puparium formation after embryo
collection. To simplify the analysis of twin-spot MARCM clones,
only female samples were used in this study. For presentation
purposes, wild-type mCD8::GFP- and rCD2::RFP-positive multi-
cellular NB clones are shown in green in all figures. Primary
antibodies used in this study include rat monoclonal antibody to
mCD8 (1:100, Invitrogen), rabbit antibody to RFP (1:500,
Clontech), and nc82 (1:100, DSHB). Secondary antibodies with
different fluorophores, Cy3 (Jackson lab), Cy5 (Jackson lab), and
Alexa 488 (Invitrogen), were used 1:200, 1:200, and 1:750 dilution
in this study. Immunofluorescent signals were collected by Zeiss
LSM 710 confocal microscopy and then processed using Adobe
GH146-positive adPNs as revealed by dual-expression-
control MARCM. LexA::GAD-GH146 and acj6-GAL4 were
utilized to label GH146-positive adPNs in magenta and all the
adPNs in green in the same NB clones. About 32 green-only
adPNs exist in the clone generated in early larvae (A) or even
within the one induced during the birth of the last GH146-positive
VA1lm-targeting adPN (B). Different focal sections of the AL are
shown underneath. Note six glomeruli (VM5d, VM5v, VC3, VC4,
DL2d, and DL2v) are exclusively labeled in green and selectively
innervated by GH146-negative adPNs. Glomerular identity in this
About 32 adPNs are made after birth of
and all other supporting figures was determined based on nc82
immunostaining (blue). The scale bar in this and all other
supporting figures equals 10 mm.
Found at: doi:10.1371/journal.pbio.1000461.s001 (3.60 MB
clone visualized with GAL4-GH146. Four focal planes shown
in (A) to (D) reveal the glomerular composition of a full-sized adPN
clone labeled by GAL4-GH146 (green). Top panels: GH146-
positive adPNs (green); middle panels: nc82 counterstaining (blue);
bottom panels: merged images.
Found at: doi:10.1371/journal.pbio.1000461.s002 (3.00 MB TIF)
The glomerular pattern of a full-sized adPN
VP3-targeting single-cell clone by acj6-GAL4. (A–D)
Glomerular targets of the entire adPN lineage are shown in four
focal planes. Top panel: labeling of all adPNs by acj6-GAL4
(green); middle panels: nc82 counterstaining (blue); bottom panels:
merged images. (E) An embryonic-born VP3-targeting adPN
shown in the regions of the AL (top and middle panels) and the LH
(bottom panel). Top and bottom panels: composite confocal
images; middle panel: a single focal section.
Found at: doi:10.1371/journal.pbio.1000461.s003 (4.72 MB TIF)
Labeling of a full-sized adPN NB clone and a
single-cell clones of adPNs. Mosaic brains carrying differen-
tially marked single-cell clones of adPNs. Three examples are
shown for each specific pair. Note co-migration of neurites in
sibling neurons targeting the same glomerulus (A–F). In contrast,
distinct paths were taken by sibling neurons that target different
AL glomeruli (G, H). It is true even among PNs that have all
established a fork-like trajectory (compare [C] and [E] with [H]).
In addition, among PNs targeting the same glomerulus (A–F), the
detailed trajectories may deviate more between different brains
than within a given brain. This might reflect developmental and/
or functional plasticity of the brain.
Found at: doi:10.1371/journal.pbio.1000461.s004 (2.35 MB TIF)
Axon projections of differentially marked
born adPNs. Axon trajectories of previously unidentified
embryonic-born adPNs are shown in three different brains for
each type. Islets reveal the axons of both single adPNs (magenta)
and the accompanying NB clones (green). Note acquisition of
analogous projections among adPNs targeting the same glomer-
ulus, including the VM3-targeting adPNs (G and H) that were
born in separate windows.
Found at: doi:10.1371/journal.pbio.1000461.s005 (2.63 MB TIF)
Stereotyped axon projections of embryonic-
Found at: doi:10.1371/journal.pbio.1000461.s006 (0.24 MB
Embryonic-born twin-spot MARCM clones
Found at: doi:10.1371/journal.pbio.1000461.s007 (0.25 MB
Embryonic-born twin-spot MARCM clones
spot MARCM clones using GAL4-GH146.
Found at: doi:10.1371/journal.pbio.1000461.s008 (0.17 MB
Distribution of cell numbers in adPN twin-
spot MARCM clones using acj6-GAL4.
Found at: doi:10.1371/journal.pbio.1000461.s009 (0.12 MB
Distribution of cell numbers in adPN twin-
Comprehensive Lineage Analysis by Twin-Spot MARCM
PLoS Biology | www.plosbiology.org 10August 2010 | Volume 8 | Issue 8 | e1000461
Found at: doi:10.1371/journal.pbio.1000461.s010 (0.15 MB
Branch number of different adPN types in the
We thank Jon-Michael Knapp for the assistance on Figure 1D and Jon-
Michael Knapp, Alexander G. Vaughan, and Dr. Gregory S. X. E. Jefferis
for critical reading of the manuscript. We thank members of the Lee lab for
helpful discussions through the entire project. We also thank Crystal
Sullivan for administrative support.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: HHY CFK TL.
Performed the experiments: HHY CFK YH PD JCK. Analyzed the data:
HHY CFK. Wrote the paper: HHY TL.
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