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: email@example.com
. 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
PLoS Biology | www.plosbiology.org1 August 2010 | Volume 8 | Issue 8 | e1000461
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.
Comprehensive Lineage Analysis by Twin-Spot MARCM
PLoS Biology | www.plosbiology.org2August 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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Comprehensive Lineage Analysis by Twin-Spot MARCM
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