Tools for neuroanatomy and neurogenetics
Barret D. Pfeiffer*, Arnim Jenett*, Ann S. Hammonds†‡, Teri-T B. Ngo*, Sima Misra‡, Christine Murphy*, Audra Scully§,
Joseph W. Carlson†, Kenneth H. Wan†, Todd R. Laverty*, Chris Mungall§, Rob Svirskas*, James T. Kadonaga¶,
Chris Q. Doe?, Michael B. Eisen‡**, Susan E. Celniker†, and Gerald M. Rubin*§††
*Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn VA 20147;†Department of Genome and Computational Biology, Life Sciences
Division, and **Department of Genome Sciences, Genomics Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720;‡Department of Molecular
and Cellular Biology and§Howard Hughes Medical Institute, University of California, Berkeley CA 94720;¶Section of Molecular Biology, University of
California, San Diego, La Jolla, CA 92093; and?Institutes of Neuroscience and Molecular Biology, Howard Hughes Medical Institute, University of Oregon,
Eugene OR 97403
Contributed by Gerald M. Rubin, April 17, 2008 (sent for review March 13, 2008)
We demonstrate the feasibility of generating thousands of trans-
genic Drosophila melanogaster lines in which the expression of an
exogenous gene is reproducibly directed to distinct small subsets
of cells in the adult brain. We expect the expression patterns
produced by the collection of 5,000 lines that we are currently
generating to encompass all neurons in the brain in a variety of
intersecting patterns. Overlapping 3-kb DNA fragments from the
flanking noncoding and intronic regions of genes thought to have
patterned expression in the adult brain were inserted into a
defined genomic location by site-specific recombination. These
fragments were then assayed for their ability to function as
transcriptional enhancers in conjunction with a synthetic core
promoter designed to work with a wide variety of enhancer types.
An analysis of 44 fragments from four genes found that >80%
drive expression patterns in the brain; the observed patterns were,
on average, comprised of <100 cells. Our results suggest that the
D. melanogaster genome contains >50,000 enhancers and that
multiple enhancers drive distinct subsets of expression of a gene in
each tissue and developmental stage. We expect that these lines
will be valuable tools for neuroanatomy as well as for the eluci-
dation of neuronal circuits and information flow in the fly brain.
enhancer ? gene expression ? promoter ? transcription ? transgenic
cells. Consequently, the classic genetic methods that have been
so powerful in elucidating embryonic development and other
processes in Drosophila melanogaster are not adequate to probe
the function of the nervous system (1). Instead, we will need to
be able to assay and manipulate the function of individual
neurons with the same facility as we can now assay and manip-
ulate the function of individual genes.
A variety of genetically encoded probes have been developed
that allow researchers to visualize individual neurons to study
anatomy, as well as to monitor and modulate the activity of
neurons to study physiology and behavior. The utility of these
probes is highly dependent on the precision with which their
expression can be directed to small subsets of neurons in
reproducible, controllable, and convenient ways. The primary
objective of the work described in this report was to expand the
tools available to accomplish such precise, controlled expression
in the nervous system of D. melanogaster.
Researchers have known for more than 20 years how to
identify and, to some extent, manipulate the promoters and
enhancers that control the temporal and spatial expression of
individual genes in Drosophila (2). This work, and similar studies
in other animals, has revealed that the complex spatial and
temporal expression pattern of a gene usually results from the
combined action of a set of individual enhancer elements that
act, in a largely autonomous manner, to dictate aspects of the
expression of that gene (3, 4). The number of enhancers per gene
he functional elements of the nervous system and the
neuronal circuits that process information are not genes but
varies widely but is generally thought to be in the range of 2 to
10 in Drosophila (5).
Because individual enhancers appear to represent the funda-
mental cis-acting modules through which gene expression pat-
terns are generated, our objective was to identify a large set of
enhancers that could each reproducibly drive expression of a
reporter gene in a distinct, small subset of cells in the adult CNS.
Ideally, the number of defined expression patterns should be
large enough that, in sum, they would cover the entire brain
several times over in a variety of overlapping patterns.
The feasibility of this approach depends on a number of
factors. First, enhancers from a wide range of genes whose core
promoters contain different sequence motifs must each function
robustly when placed in a defined genomic location with a
common core promoter. Second, the expression pattern driven
by a given enhancer must be highly reproducible from animal to
animal. Third, the expression patterns driven by individual
enhancers should contain an appropriately small fraction of the
cells in the brain to make them useful tools for neuroanatomy
and behavioral genetics. Finally, the methods for transgenesis
and for identifying suitable enhancers must be efficient enough
to permit the generation of the required thousands of transgenic
lines. Here, we report the development of a strategy that we
believe meets all four of these criteria.
Results and Discussion
Overview of Experimental Strategy. We selected 925 genes for
which available expression data or predicted function implied
expression in a subset of cells in the adult brain, for example,
genes encoding transcription factors, neuropeptides, ion chan-
nels, transporters, and receptors [supporting information (SI)
intergenic regions of these genes, as well as any of their introns
larger than 300 bp, with fragments of DNA that averaged 3 kb
in length and overlapped (in regions that could not be covered
by a single fragment) by ?1 kb. The fragments were generated
by PCR from genomic DNA using primers designed to lie in
areas of low evolutionary conservation to minimize disruption of
individual enhancers. Because the average size of an enhancer
element is only a few hundred base pairs (5), we expected that
Author contributions: B.D.P., S.E.C. and G.M.R. designed research; B.D.P., A.J., A.S.H.,
the paper. A.J. analyzed the brain images; S.M., J.W.C., C. Mungall, R.S., and M.B.E.
contributed informatics methods and analyses; and J.T.K. designed the core promoter.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
††To whom correspondence should be addressed at: Janelia Farm Research Campus,
Howard Hughes Medical Institute, Ashburn, VA 20147. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
July 15, 2008 ?
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nearly all enhancers would be intact in at least one fragment.
This process generated 5,200 fragments that were cloned, se-
quence-verified, and inserted upstream of a core promoter (Fig.
1A). In ?200 cases in which the upstream intergenic region was
small, we generated PCR fragments that also contained the start
site of transcription and used them to create transcriptional
Enhancer activity could be tested by imaging the expression
patterns that these fragments produce in transgenic animals. In
the experiments described here, each enhancer drives the ex-
pression of the yeast transcription factor GAL4 (6, 7). We
detected the expression of GAL4 either directly by whole mount
in situ hybridization to its mRNA or by the ability of GAL4
products were then detected by immunocytochemistry and con-
focal microscopy of whole mount tissue (8).
Drosophila core promoters are ?80 bp and contain the start
site of transcription. An enhancer requires the presence of
specific sequence motifs in the core promoter to function
properly and the core promoters of different genes vary in their
content of these motifs; thus, not all enhancers function effi-
ciently with all core promoters (9). It has been shown that potent
core promoters can be created by the incorporation of multiple
core promoter motifs into a single promoter (10); more impor-
tantly, such promoters would be expected to respond to a wider
range of enhancers than naturally occurring core promoters. To
assay the enhancer elements from many different genes by using
a single core promoter, we constructed a Drosophila synthetic
core promoter (DSCP) that contains the TATA, Inr, MTE, and
DPE sequence motifs (Fig. 1B).
We used the phiC31 site-specific integration system (11) to
location. We selected an integration site, attP2 (Fig. S1) (11),
which allows high levels of expression but does not appear to
strongly influence the observed pattern of expression of the
inserted construct; when inserted at this site, constructs that
carry the DSCP but no enhancer lack detectable adult CNS
expression (data not shown), many DNA fragments fail to drive
any CNS expression, and there are no common pattern elements
shared across large numbers of lines that do show CNS expres-
sion. Because of the consistent nature of the integration site, we
could reliably compare the patterns of expression generated by
different enhancer sequences and, once the expression pattern
was determined, have confidence that we could drive the ex-
pression of other reporter genes in that pattern. Finally, having
all constructs inserted at the same genomic location greatly
simplifies subsequent genetic manipulations.
Evaluation of a Drosophila Synthetic Core Promoter. We compared
the expression patterns driven by 40 fragments derived from the
dachshund (dac), earmuff (CG31670), and twin of eyeless (toy)
genes when the fragments were paired either with their cognate
promoter or with the DSCP (Tables S1–S3); the genomic extents
of these fragments are shown as blue bars in Figs. 2A, 3A, and
4A. The dac, earmuff, and toy genes encode evolutionarily
conserved transcription factors, with their vertebrate homologs
being Dach, Fezl, and Pax6, respectively. An important feature
of these genes for our purposes was that they were annotated as
having unique transcription start sites, allowing us to select a
single endogenous promoter for each.
To compare expression patterns, we established a controlled
vocabulary for annotating patterns of axonal and dendritic
projections. We first divided the brain into 45 identifiable brain
structures, for example, antennal lobe, ellipsoid body, or great
commissure. We separately scored each of these regions by using
a zero to five scale for three parameters: intensity, distribution,
and shape. (See SI Methods for a complete description of the
controlled vocabulary.) Based on this scoring, we found that the
variation between patterns generated with the two promoters
was only slightly higher than that seen when comparing the same
construct in multiple animals (see below); for 85% of the
fragments, the patterns they drove when paired with the DSCP
or their cognate promoter were identical in all three parameters
in each brain structure. The patterns observed were larger or
more pronounced in 10% of the cases with the DSCP and in 5%
of the cases with the cognate promoter (see for example Fig. 2
B–E and G–J); however, even these differences were very subtle.
In the embryo, we found that the DSCP routinely drove stronger
expression (Fig. 3B). These results indicate that, for our pur-
poses, the DSCP serves as an adequate surrogate for the core
promoters of individual genes.
Relationship of the Expression Patterns Driven by Individual Frag-
ments to the Expression Pattern of the Gene. Our expectation from
previous work was that individual fragments would drive subsets
of the endogenous expression pattern of a gene (2–5). However,
it was also likely that individual fragments, when taken out of
context and freed from negatively acting elements, as well as
from the necessity to compete with other enhancers for access
to the core promoter, would drive expression in cells where the
endogenous gene was not expressed. To address this possibility,
3B) and toy (Fig. 4 B–D) with the patterns driven by individual
fragments of these genes when combined with the DSCP. As
expected, we found that individual fragments generally drove
to be able to reproduce all of the components of that pattern.
However, with some of the fragments, we also saw reproducible
expression in cells that do not express the endogenous gene.
The specificity and reproducibility of the patterns driven by
individual fragments were illustrated by the expression pat-
terns driven by six different fragments derived from the toy
gene within the embryonic CNS (Fig. 4 C and D and Fig. S2).
Endogenous toy protein was expressed in a highly stereotyped
1. The fragment of genomic DNA to be tested for enhancer
activity is generated by PCR, cloned into a Gateway donor
vector and its identity verified by DNA sequencing.
2. Site-specific recombination is used to transfer the
fragment into the integration vector pBPGUw.
3. Site-specific integration using PhiC31 recombinase is used
to place each test construct in the same genomic location.
4. Lines of homozygous integrants are maintained and are crossed to
appropriate UAS-GFP lines for assaying expression.
enhancer activity. (A) Diagram of the vectors and sequential cloning steps. (B)
Sequence of the Drosophila synthetic core promoter (DSCP). Sequences high-
lighted in yellow were added to the promoter of the eve gene. The positions
of known promoter motifs are indicated.
Strategy for constructing transgenic lines to test DNA segments for
www.pnas.org?cgi?doi?10.1073?pnas.0803697105Pfeiffer et al.
pattern of neurons within the stage 16 CNS: a pair of toy medial
(TM) neurons, a superficial neuron cluster, a toy intermediate
(TI) neuron cluster, and three toy deep lateral (TDL) neurons.
Each fragment drove reproducible expression in a subset of the
endogenous toy-positive neurons; for example, R9H03 was the
only line expressed in the pair of TM neurons, whereas R9G10
was the only line expressed in all three TDL neurons. Each line
also showed unique but reproducible expression in a subset of
neurons that do not express toy protein; for example, R9G09
and R9G10 were the only toy-derived lines expressed in the
RP2 motor neuron, whereas R9G09 and R9H01 were the only
lines expressed in the U motor neurons (data not shown). We
conclude that each of these fragments contains sequences that
drive expression in a different, reproducible subset of the
native toy pattern; in addition, when taken out of context, they
also drive expression within distinct, reproducible subsets of
neurons that do not normally express toy.
Total Expression Pattern. We tested 44 fragments derived from
genes encoding the transcription factors Earmuff, TOY, DAC,
and the G protein-coupled receptor octopamine receptor 2 (Fig.
of these fragments generated expression patterns comprising 3
to 1,000 cells in the adult central brain; the central brain
corresponds to the brain minus the optic lobes. The mean
number of cells showing detectable expression was 95 in these
lines; the median number of expressing cells was only 19. These
cell numbers were much smaller than observed in a random
sample of 27 enhancer trap lines where the observed mean and
median were 370 and 180, respectively (Fig. 6); this sample was
consistent with the expression patterns generally seen with
enhancer trap lines (8). In enhancer trap lines, a transposon
carrying a core promoter and a reporter gene is inserted
randomly in the genome; the broader expression observed is
likely a consequence of individual enhancer trap lines reporting
the influence of multiple enhancers.
The patterns driven by a particular fragment are highly
dynamic during development. For example, compare fragment
R9D11 in the late larva (Fig. 3F) and the adult (Fig. 3J). The
larva showed strong expression in ?5% of the secondary lin-
eages that produce the cells of the adult central brain, but in the
adult central brain, expression is limited to approximately a
Further subdivision of the fragments will be required to
determine the extent to which distinct enhancer activities within
would represent the expression pattern of a single enhancer.
Overlapping fragments often showed overlapping patterns, sug-
gesting that further subdivision would be possible. For example,
compare the patterns driven by the fragments R9G08, R9G09,
and R9G10, which drove expression in the TI cluster of embry-
onic TOY-expressing neurons; R9G08 and R9G10 drove ex-
pression in distinct subsets, whereas R9G09 drove expression in
most or all of the TI neurons (see Fig. 4D and Fig. S2).
The Patterns Generated by the Same Enhancer in the Adult Brains of
Different Animals Are Highly Reproducible. If the GAL4-expressing
lines we created were to have maximum utility, the patterns they
produced would have to be highly reproducible from animal to
animal. Variability might result from stochastic variation in gene
expression activation or in anatomical variation. The degree of
variability of adult brain anatomy between individual adult flies
of the same genetic makeup has not been well documented. We
compared the patterns generated by individual fragments in
scheme described above and blind study conditions, 95% of the
isogenic brains from different animals were scored with identical
annotations; even in the 5% that were not scored identically, the
differences were subtle. This suggests that variation among
animals at the granularity that we were scoring was minimal;
however, we did observe that the positions of cell bodies vary
much more than arborization patterns. This is illustrated in Fig.
Chromosome arm 2L
chromosomal region surrounding the gene showing the structures of the transcription unit and those of adjacent genes (data taken from D. melanogaster
R62A03 (green) is a promoter fusion, and we did not obtain data for R9C06 (gray). Total (B–E and G–J) or partial projections (F) of confocal images of the brain
(B, C, F–J) or ventral nerve cord (D and E) of 2- to 5-day old adults of the indicated transgenic line. Fragments were tested with either the DSCP (B, D, F, G, and
is counterstained in magenta (see SI Methods for details). Embryonic expression patterns are shown in Fig. S3. The gray scale images show only the
Testing fragments from the dac genetic region for enhancer activity with both the DSCP and the endogenous dac promoter. (A) Diagram of the
Pfeiffer et al.
July 15, 2008 ?
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3O, which shows the expression pattern of line R9D11 in the
fan-shaped body of four animals.
to establish a collection of transgenic Drosophila lines, each
directing expression to a small subset of cells in the adult brain,
and that, in sum, would cover all cells in the brain. More than
80% of the 44 fragments we tested from four genes gave
expression in the adult brain, suggesting that we would generate
?4,000 lines with patterns from the analysis of our initial 5,200
putative enhancers. More than half of the fragments we tested
gave expression in 10 to 200 cells: 0.03–0.7% of the 30,000 cells
estimated to comprise the central brain (W. Pereanu, personal
communication). We believe this fraction of cells is a useful
number for anatomical, physiological, and behavioral studies.
We recognize that the preferred cell number for behavioral
studies is not known and will certainly depend on the particular
behavior and assay; however, we expect that our lines, singly or
in combination, will provide the versatility needed to generate
expression patterns of the desired sizes. It is possible that the
2,000 lines we expect to generate having expression patterns
within the 10 to 200 range of cell numbers will be adequate to
1950 kb 1960 kb
Chromosome arm 2L
R9D03 R9D06R9D10 R9D11
R9D03R9D04 R9D06R9D11 R9D10R9D09R9D08R9D05
earmuff gene in embryonic stages 4–6, 7–8, 9–10, 11–12, and 13–16 visualized by whole mount in situ hybridization with a probe to earmuff mRNA shown
adjacent to the expression produced by the fragments R9D03, R9D04, R9D05, R9D08, R9D09, R9D10, and R9D11 when placed in the enhancer test vector and
fragment R9D06 as a promoter fusion. Transgene expression is visualized by whole mount in situ hybridization with a probe to GAL4 mRNA. The enhancer
constructs shown use the DSCP; for stages 9–10, we also show data obtained with the endogenous earmuff promoter. Dorsal views are shown except for stages
4–6, where a lateral view is also shown below the dorsal view; anterior is at Left. (C, D, E, and F) Expression driven by the indicated fragment in late third instar
larvae. The clusters of labeled cells seen in F represent distinct lineages of secondary neurons; this labeling is not maintained in the adult (J). (G–O) Expression
in the adult brain of the indicated lines. A total projection (K) and single optical section (L) of the optic lobes of the brain shown in G. (O) Expression in the
fan-shaped body of line R9D11 in four different brains.
Patterns generated by fragments of the earmuff gene in embryos, larvae, and adults. (A) The genomic map of the earmuff locus. (B) Expression of the
www.pnas.org?cgi?doi?10.1073?pnas.0803697105Pfeiffer et al.
represent all cells in the central brain in a variety of overlapping
patterns. If not, it is straightforward to generate additional lines.
Our results reveal several features of genome organization.
The number of distinct patterns we observed implies that there
are likely to be ?50,000 transcriptional enhancers in the Dro-
sophila genome; ?80% of the fragments we tested showed
enhancer activity, it would take ?50,000 such fragments to cover
the entire genome, and many of these fragments are likely to
carry multiple enhancers. A gene that is expressed in many
tissues and developmental stages might have a single enhancer
that controls all aspects of expression in a given stage or tissue;
for example, an ‘‘adult brain enhancer’’. Alternatively, the
expression pattern at each stage and tissue might be generated
by the sum of the actions of multiple enhancers, each controlling
a subset of the pattern. Our data strongly favor the latter
possibility. Furthermore, our results suggest that enhancers are
reused throughout development, although the resolution of our
current experiments was not sufficient to distinguish two closely
linked enhancers from a single enhancer. It is also clear that
enhancers, taken out of context, in addition to driving a subset of
the expression pattern of the endogenous gene, often show highly
stereotyped expression that is not displayed by the endogenous
gene; this expression may reflect either the absence of competition
between enhancers or the separation from repressive elements.
We believe that the lines we have generated, where a molec-
ularly defined DNA fragment drives expression, have several
map of the toy locus and the positions of the tested fragments. (B) Expression of the endogenous toy mRNA and the expression of GAL4 mRNA driven by the
indicated nine fragments shown in stage 13–16 embryos; the other nine fragments shown in A did not drive detectable expression at this stage. Dorsal (Upper)
and lateral (Lower) views are shown; anterior is at Left. (C) Endogenous toy protein (magenta in the merged image) and nuclear localized GFP (green in the
merged image) expression driven by the R1A02 fragment in an abdominal CNS hemisegment of a stage 16 embryo (anterior, up; midline, dashed line). Three
toy-positive neurons and the subset of toy-positive neurons in which each indicated fragment drives expression (deep neurons, blue; intermediate neurons,
green; superficial neurons, orange); each fragment also drives expression in a reproducible set of toy-negative neurons that are not shown in these diagrams.
in the brain (E and G) or ventral nerve cord (F and H) of lines R9G08 (E and F) and R1A02 (G and H).
Chromosome arm 3R
18330 kb 18340 kb
octopamine receptor 2
R20E11 R20C11 R19H07
the indicated fragments in the adult brain and ventral nerve cord.
Distinct expression patterns generated by fragments of the octopamine receptor 2 gene. (A) Diagram of the genomic locus. (B–D) Expression driven by
Pfeiffer et al.
July 15, 2008 ?
vol. 105 ?
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advantages over existing tools for neuroanatomy and neuroge-
netics. The patterns we observed were less broad than those
observed in enhancer trap lines, and our constructs were all at
the same genomic location, facilitating subsequent genetic ma-
nipulations. It is also straightforward to attempt to produce
smaller patterns by subdividing the fragments; these fragments
were each large enough to carry several distinct enhancers. Most
importantly, because our constructs were all inserted at precisely
the same genomic location, the effects of local genomic envi-
ronment on expression could be held constant. Thus, we should
be able to change the gene whose expression is driven by a
particular enhancer and have confidence that the expression
pattern would remain the same. In this way, we could readily
produce lines that drive, instead of GAL4, the expression of
other transcription factors such as LexA (12), inhibitors of
transcription factor function such as GAL80 (13), or recombi-
nases such as Flp (7, 14) in the same patterns that we have
determined for GAL4.
The small number of cells labeled in our lines can facilitate
anatomical analysis, and the imaging of the expression patterns
of several thousand such lines will provide a good overall view
of the morphological range of neurons present in the fly brain
and their projection patterns. Such studies can be aided by
expression of markers directed to axons, dendrites, or synapses
(15, 16). Stochastic labeling of individual cells within these
patterns can be accomplished by using recombinases to facilitate
detailed anatomy of individual cells (13, 14). It will be informa-
tive to examine the extent to which the cells that express a given
enhancer share developmental, physiological, or functional
properties and how shared activation of a given enhancer is
related to the concept of cell type.
Finally, these lines will provide the ability to express geneti-
cally encoded indicators of function (17) or modifiers of neu-
ronal activity (18) in well defined small subsets of neurons. We
are optimistic that learning how many different behaviors are
modified when the function of each of these small cell popula-
tions is altered will provide useful insights into the organization
of neuronal circuits and information flow within the fly brain.
Standard molecular and histochemical methods were used; details of the
constructs and protocols are given in SI Methods. Vectors are available from
Soo Park provided technical assistance in the molecular biological aspects of
this work; Richard Weiszmann and Amy Beaton assisted with in situ hybrid-
Megan Hong, and Monti Mercer assisted with genetics and stock mainte-
Medical Institute, Ashburn, VA) provided media and general laboratory sup-
port. Eric Trautman (Janelia Farm Research Campus, Howard Hughes Medical
Institute, Ashburn, VA) provided additional informatics tools. Phuong Chung
and Julie Simpson taught us brain dissection and histochemical methods;
work; and Susan Zusman and Michael Tworoger of Genetic Services, Inc.,
generated most of the transgenic lines. We thank Michael Layden for help in
generating the data shown in Fig. 4C and Fig. S2. This work was supported by
the Howard Hughes Medical Institute and by National Institutes of Health
Grants GM076655–01A1 (to S.E.C.) and GM041249 (to J.T.K.).
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cells than those found in enhancer trap lines. The gray bars in the histogram
show the number of cells found in the patterns within the central brain of the
3A (earmuff), 4A (toy), and 5A (octopamine receptor 2) genes. The black bars
show the number of cells found in 27 enhancer trap lines that were chosen
randomly from the unpublished collection of J. Simpson and B. Ganetsky and
are typical of the type of patterns seen in other enhancer trap collections (8).
The patterns driven by individual fragments generally contain fewer
www.pnas.org?cgi?doi?10.1073?pnas.0803697105 Pfeiffer et al.